(August 2003)

As early as the 19th century it was known that phosphates could be bound to proteins. Most examples of these ‘phosphoproteins’ were found in milk (caseins) and egg yolk (phosvitin) and were simply considered a biological method of providing phosphorus as a nutrient. Therefore, the existence of phosphoproteins was considered a consequence of metabolic reactions, and nothing more, for almost a century after their discovery [1].

In the 1950’s this all began to change as phosphoproteins began to emerge as key regulators of cellular life. An initiating factor of this emergence occurred in 1954, when an enzyme activity was observed that transferred a phosphate onto another protein [2] -a biological reaction called phosphorylation. The protein responsible was a liver enzyme that catalyzed the phosphorylation of casein and became known as a protein kinase (see Figure 1), the first of its kind to be discovered. A year later, the role of phosphorylation became more interesting as Fischer and Krebs [3], and Wosilait and Sutherland4, showed that an enzyme involved in glycogen metabolism was regulated by the addition or removal of a phosphate, suggesting that reversible phosphorylation could control enzyme activity. This idea was later proven to be true and has now seeped into virtually every aspect of cell biology.

Today, it is thought that one third of the proteins present in a typical mammalian cell are covalently bound to phosphate (i.e. they are phosphorylated at one time or another). The study of cell biology is now littered with examples of regulation by phosphorylation: increasing or decreasing the biological activity of an enzyme, helping move proteins between subcellular compartments, allowing interactions between proteins to occur, as well as labeling proteins for degradation. The variety is immense and now many human diseases have been recognized to be associated with the abnormal phosphorylation of cellular proteins.

These developments have brought the study of phosphorylation into the limelight of medical research, a fact that was recognized in 1992 when Fischer and Krebs received the Nobel Prize in medicine for their pioneering efforts. For those interested, the lectures given by Fischer and Krebs upon receiving the Noble Prize are an excellent overview of the early days of this field [5].

How can phosphorylation control enzyme activity?

Phosphorylation refers to the addition of a phosphate to one of the amino acid side chains of a protein. Remember that proteins are composed of amino acids bound together and that each amino acid contains a particular side chain, which distinguishes it from other amino acids. Phosphates are negatively charged (with each phosphate group carrying two negative charges) so that their addition to a protein will change the characteristics of the protein. This change is often a conformational one, causing the protein to change how it is structured (see Figure 2).

This reaction is reversible by a process called dephosphorylation. The protein switches back to its original conformation when the phosphorus is removed (see Figure 2). If these two conformations provide the protein with different activities (i.e. being enzymatically active in one conformation but not the other), phosphorylation of the protein will act as a molecular switch, turning the activity on or off.

The transfer of phosphates onto proteins is catalyzed by a variety of enzymes in the cell. Although the variety is large, all of these enzymes share certain characteristics and fall into one class of proteins, called protein kinases [6]. Their similarities stem from the group’s ability to take a phosphate off the chemical energy-carrying molecule ATP and place it onto an amino acid side chain of a protein (see Figure 3). The hydroxyl groups (-OH) of serine, threonine, tyrosine or histidine amino acid side chains are the most common target. A second class of enzymes is responsible for the reverse reaction, in which phosphates are removed from a protein. These are termed protein phosphatases.

The use of the phosphorylation/dephosphorylation of a protein as a control mechanism has many advantages:

• It is rapid, taking as little as a few seconds.
• It does not require new proteins to be made or degraded.
• It is easily reversible.

The extensive use of this control mechanism is apparent by the large number of known kinases and phosphatases [6]. Even in a simple organism like yeast, approximately 3 percent of its proteins are kinases or phosphatases. Some of these enzymes are extremely specific, potentially phosphorylating or dephosphorylating only a few target proteins, while others are able to act broadly on many proteins. The examples of known targets of phosphorylation include most protein components of the cell, including enzymes, structural proteins, cell receptors, ion channels and signaling molecules. If a protein is controlled by its phosphorylation state, its activity at any one time will be directly dependent on the activity of the kinases and phosphatases that act on it. It is quite common for a phosphate group to be added or removed from a protein continually, a cycle that allows a protein to switch rapidly from one state to another.

External Signals can activate protein kinases and phosphatases

The reception of a signal on the surface of a cell often results in the activation of kinases and phosphatases [6]. Once activated, cellular phosphorylation patterns will begin to change, with various proteins being phosphorylated or dephosphorylated. The final result will be various changes in cellular behavior.

Many of the proteins that are phosphorylated upon reception of a signal are protein kinases as well. This organization of kinases produces a phosphorylation cascade [6] (see Figure 4), in which one protein kinase is activated by phosphorylation upon reception of a signal, this kinase then phosphorylates the next kinase in the cascade, and so on until the signal is transmitted through the cell. In such a system, the kinase cascade can start with the receptor itself (which is often a kinase) or a free-floating cytoplasmic kinase. Upon reception of a signal, these phosphorylation cascades continue to function until protein phosphatases are activated and shut off their transmission.

In animal cells, these cascades are mediated by two types of kinases: serine/threonine kinases [6] (which phosphorylate serine and threonine amino acid side chains) and tyrosine kinases [6] (which phosphorylate tyrosine amino acid side chains).

Phosphorylation in response to a signal produces a second outcome apart from the activation of kinases and phosphatases, which involves the production of binding sites for proteins to interact [6]. This process is different from the activation of a protein by phosphorylation (in which the addition of a phosphate causes differences in enzyme activity), since it does not necessarily change the inherent activity of the molecule that has been phosphorylated. Instead, it creates a phosphorylated amino acid on the molecule that another protein can bind to (see Figure 4). Upon reception of a signal, some membrane-bound receptors will become tyrosine phosphorylated. Free-floating proteins then bind to these phosphotyrosine sites and are thus concentrated near the receptor. This concentration often leads to the activation of additional proteins by bringing together molecules that normally would not be in close proximity.

Through the use of phosphorylation cycles and cascades, the cell is able to regulate a diverse set of processes, including cellular movement, reproduction and metabolism. It is the simplicity, reversibility and flexibility of phosphorylation that explains why it has been adopted as the most general control mechanism of the cell.

Addition Reading And Texts Consulted

1. Pawson T. 1994. Introduction: Protein Kinases. FASEB J. 8:1112-1113.

2. Hardie DG, ed. 1999. Protein Phosphorylation: A Practical Approach. Oxford/New York: Oxford University Press. 431p.

3. Hardie DG, Hanks S, eds. 1995. The Protein Kinase Factsbook. London/San Diego: Academic Press. Vol. 1-2.

References

1. Marks F, ed. 1996. Protein Phosphorylation. Weinheim: VCH.

2. Burnett G, Kennedy EP. 1954. J. Biol. Chem. 211: 969-980.

3. Fischer EH, Krebs EG. 1955. J. Biol. Chem. 216: 121-132.

4. Sutherland EW, Wosilait WD. 1955. Nature 175: 169-170.

5. Nobel e Museum: Nobel Prize in Physiology or Medicine (1992):

6. Sefton BM, Hunter T. 1998. Protein Phosphorylation. San Diego: Academic Press.

(Art by Jane Wang – note that high res versions of image files available here)

(August 2003)

In the beginning, there were chickens

Surprisingly enough, our understanding of tumour formation has its roots not in humans, but in the chicken. For the past century it has been known that viruses can be causative agents of cancer. Cancer-causing elements were first described in viruses infecting poultry in 1909 [1]. Later, it was shown that the injection of these viruses was sufficient for tumour formation. But the molecular basis of cancer was unearthed only with the discovery of cellular homologues to these cancer-causing elements in 1976 [1]. When the viral DNA sequences were isolated and characterized they were found to have high similarity to genes that already existed in the animal. These genes were named proto-oncogenes, and their viral, cancer-causing counterparts were called oncogenes.

Discovery of similar viral and cellular DNA sequences inspired a comparison of the two, revealing particular mutational differences. The hypothesis that the virus had at one point incorporated host genomic DNA into its own genome was suggested. This hypothesis postulated that these DNA sequences encoded for proteins involved in cell proliferation. By incorporating these genes and mutating them, the virus increased host cell proliferation, and consequently, its own replicative potential. This hypothesis led to the prediction that mutated forms of these same genes would likely be found in naturally occurring tumour cells as well. Scientists examined tumour cells for such genes, and transfected tumour DNA into normal fibroblasts to determine if the introduction of these mutated genes was sufficient to cause cancer. Indeed, they were, and awareness of the cellular oncogene was born [1].

Change a proto-oncogene to an oncogene and voila!

Oncogenes are mutated genes that contribute to cancer development by disrupting a cell’s ability to control its own growth and DNA repair mechanisms2,5. Normally mitosis (cell division) is a carefully regulated event, requiring the activation of one protein to activate another, in what is known as a signal transduction cascade. This cascade eventually culminates in changes in gene expression that prepares the cell for mitotic events.

Oncogenes are mutated forms of proto-oncogenes; these genes are often involved in growth signalling and anti-apoptotic pathways [2,5]. When proto-oncogenes mutate to become oncogenes they retain their functionality, but are no longer capable of responding to normal regulatory signals (see Figure 1).

Such is the case of the growth factor receptor egfr (epidermal growth factor receptor). In its proto-oncogenic form it requires binding of the growth factor molecule to enable its kinase activity [3]. The catalytic domain then proceeds to transfer phosphate groups to its target proteins to activate them, which ultimately leads to translation of proteins involved in mitosis. The oncogenic form of egfr produces a receptor that does not require binding of growth factor, but instead is constitutively active (see Figure 2). In this way the oncogene product is capable of always activating the pro-growth pathway in the absence of pro-growth signals. Also, oncogenic mutations are dominant because only one mutated allele is necessary to confer the cancerous behavior [5]. At present, over 100 oncogenes have been identified in various cancers [2].

There are several ways in which oncogenes can promote the formation of a tumour. A cell’s growth is limited in two ways: 1) inactivation of the growth pathway and 2) activation of the apoptotic or differentiation pathways [6]. A tumour is only able to develop once it has found a means to bypass these two pathways. Hence, oncogenes are typically mutated forms of regulatory proteins within these pathways. This includes over-production of a growth factor, constitutively active receptors or intermediary molecules, or increased expression of proteins that inhibit apoptosis. No one oncogene itself results in cancer – cancer requires several mutations [6]. But one oncogene can increase cell division, and consequently, increase the probability of other mutations occurring, which can eventually culminate in cancer.

How does a proto-oncogene become an oncogene?

An oncogene can be the result of several different types of DNA alterations. These fall into two broad categores: point mutations and chromosomal rearrangements. Point mutation refers to the substitution of one base for another, resulting in a change in that particular amino acid. In contrast, chromosomal rearrangements involve whole pieces of chromosomes breaking off and fusing with other chromosomal fragments. Both of these events can result in either altered activity or intracellular levels of the protein (see Figure 3).

Point mutations most commonly produce a protein that has lost its ability to be regulated by external signalling, as is the case of egfr. The point mutation translates into a changed amino acid that has different biochemical properties than the amino acid the DNA sequence originally encoded for. This change in amino acid can mimic phosphorylation, or inhibit binding of negative regulatory molecules, resulting in a constitutively active protein [5].

Chromosomal translocations can produce oncogenes in several ways. Firstly, the fusion of one chromosome to another can result in a strong promoter being placed upstream of a gene that would normally be either absent or present only in very low quantities. This has the effect of increasing the intracellular concentrations and activating pathways that would normally be silenced.

Secondly, chromosomal translocations can result in fusion proteins, or proteins that are the product of two separate genes that have been joined. Chromosomes often endure a break in their coding regions — when two chromosomal fragments fuse, the genes that the breaks occurred in become one coding sequence that is transcribed and translated as one protein. Fusion proteins can be oncogenes for a number of reasons. If the fusion protein is the product of a highly transcribed protein, then expression of the fusion protein will be high. If the second portion of the fusion protein is a molecule involved in signalling that would normally be present in very low concentrations, then its sudden increase in expression as a result of being a fusion product could increase its activity.

Conversely, if a chromosomal break in a gene for a signalling protein results in the removal of its regulatory domain, then the fusion protein will consist of a molecule with signalling capacity that has no way of being negatively regulated. Such is the case of the Bcr-Abl fusion protein. Abl is a tyrosine kinase that requires cytokine stimulation to be activated. In its monomeric form Abl is inactive [4,5]. When cytokines are present, two monomeric Abl proteins bind one cytokine molecule – this close proximity allows the proteins to dimerize and auto-phosphorylate, resulting in activation. Conversely, the bcr gene contains a dimerization motif, but no kinase activity. When a translocation occurs between chromosome 9 (containing the abl gene) and chromosome 22 (containing the bcr gene), the fusion product contains the dimerization domain of Bcr and the kinase domain of Abl. Consequently, the fusion protein dimerizes in the absence of cytokine, resulting in a constitutively active tyrosine kinase and uncontrolled cell division.

The relevance of oncogenes…

After years of research and numerous attempts to cure cancer, there is still a gaping hole in our current understanding of the disease. Many oncogenes have been discovered within the last two decades. The role of oncogenes in oncogenesis, and how the proto-oncogene is mutated to give rise to this aberrant protein has been unearthed. But what clinical implications does our newly found knowledge have?

A greater understanding of oncogenes represents new ways to treat cancer. Identifying new oncogenes allows for a new means of diagnosing cancer. Further characterizing the activity of oncogenes provides us with new drug targets for effectively treating cancer. Eventually this reservoir of information may pave the way for gene therapy, and thus eliminate the occurrence of cancer. Each new piece of information gathered potentially provides us with another strength against this disease.

References

1. Weinberg, R.A. One Renegade Cell: How Cancer Begins. New York: Basic Science, 1998.

2. Proto-Oncogenes and Cancer

3. Carpenter, G. et al. (1991). Activation of second-messenger pathways by epidermal growth factor. In Brugge, J., Curran, T., Harlow, E. and McCormick, F. (Eds), Origins of Human Cancer (pp 255-263). New York: Cold Spring Harbor Laboratory Press.

4. Witte, O.N. et al. (1991). Role of the BCR-ABL oncogene in the pathogenesis of Philadelphia chromosome positive leukemias. In Brugge, J., Curran, T., Harlow, E. and McCormick, F. (Eds), Origins of Human Cancer (pp 521-524). New York: Cold Spring Harbor Laboratory Press.

5. Tannock, I.F. and Hill, R.P. The Basic Science of Oncology, 3rd ed. New York: McGraw Hill Companies Inc., 1998.

6. Hanahan D, Weinberg R.A. (2000). The hallmarks of cancer. Cell. 100: 57-70.

(Art by Jiang Long – note that high res versions of image files available here)

(August 2003)

Conversation in the biological world is quite natural. Even on the level of the cell, a busy broadcast of communications is occurring; a fact which has caught the attention of biologists. Today, one of the hottest areas in cell biology research is the study of ’signal transduction’.

Signal transduction is the study of how a cell communicates [1]. Every cell is able to communicate through having evolved the ability to produce, recognize, interpret and respond to signals in its environment. The word ’signals’ in this context refers to nothing more than chemical molecules that are floating around. Cells have learned to detect many of these chemicals. Their molecular detection components—produced by the genes they contain— allow the cells to converse in this chemical “language of the cell.”

When you come right down to it, this ability to communicate has allowed cells to evolve. If a cell could not receive or respond to signals from its environment, for example sensing food or predators, it would be unable to adapt its behavior, and over time, would be out competed by those that could communicate. Therefore, it does form a vital part of a cell.

Scientists studying how a cell communicates have learned some astonishing things about biology—one of the most important being that foul—ups in the process of signal transduction can result in disease [2]. Quite rightly, this observation has medical researchers determined to figure out why this happens? Research in this field is turning out information at a remarkable pace. Research findings are relevant to numerous diseases and the drugs used to treat them. As a result, medical science is intensely focusing its eye on the concepts and insights that this research is producing with the hope that it will improve our health. What is signal transduction? And why does it have scientists so excited?

Where did the term “Signal Transduction” Originate?

The term signal transduction is an umbrella term in biology. It is used to refer to a broad area of cellular biology research involving topics such as the chemical signals used by cells, how these signals are received, how a cell interprets them, and the ways in which cellular machinery can be used to respond [1].

“Signal transduction,” as a term, is quite instructive once its purpose and function are made clear. Essentially signal transduction ensures that a message can be converted from one form to another during its travels and still retain its original content. Let’s look at human communication as an illustration. Consider how a message is sent over the telephone: one person speaks into a receiver that converts the sound into an electrical signal, which can then be transmitted over great distances before being converted back into sound at its destination (see Figure 1). This process retains the original content of the message and is called signal transduction.

The signals sent by cells are far simpler than the highly complex messages used by humans. One cell—termed the signaling cell—produces a particular chemical molecule that is detected by another cell—the receiving cell—using a receptor protein that recognizes the molecule and responds specifically to it. The protein, acting as the receptor, is the first step in which the chemical signal present on the outside of the cell will be converted (transduced) to different signals inside the cell. These signals will subsequently direct cell behavior (see Figure 1). It is this conversion, a biologically evolved form of signal transduction, that is the essential element that allows a cell to communicate. Early scientists discovering this concept clearly understood its importance to cell survival and began using the term to refer to their research. Over time, it stuck.

The Components of Cellular communication

There are three basic components involved in how a cell communicates:

• The signals that are sent are a variety of inorganic and organic chemical molecules that are present in the environment in which a cell lives.
• Recognition of these signals is carried out by the second component—termed a receptor. This is usually located on the outside of the cell, receptors have an affinity for the chemical signals and bind to them specifically.
• Once bound, the third component, internal signaling molecules, transduce the original signal into cellular behavior.

Lets take a closer look at each of the three components that comprise the “language of the cell [3].”

1. What types of signals do cells send?

Cellular signals are inorganic or organic chemical molecules that are simply floating through the environment. The list of signals that have been discovered numbers in the hundreds and grows longer every day. Some examples include proteins, peptides, amino acids, nucleotides, steroids, and gases [3].

Some of these signaling molecules (such as gases) are naturally present in the environment. The nitrogen gas used by some bacteria is an example of a naturally occuring signaling molecule. Other signaling molecules are basic constituents of living matter (i.e. amino acids) or biologically produced by the cells themselves (i.e. proteins). Even with the large variety of available signals, communication between cells in multicellular organisms can be grouped into just a few general types [4] (see Figure 2):

• Endocrine signals are signals that are broadcasted over the entire organism, usually accomplished by secreting the signal into the bloodstream of an animal or the sap of a plant. Signal molecules used in this manner are called hormones, which are produced by endocrine cells in animals.
• If a signal is secreted but only diffuses locally, remaining in the neighborhood of the secreting cell, it constitutes a second type of communication termed paracrine signals. Since these signals do not travel far from their source, they are referred to as local mediators. Many of these local mediators are responsible for regulating inflammation at sites of infection.
• Neuronal signals are a third method and are exemplified by the neurons in our brains, which send signals over private channels to individual cells. This type of signaling is performed by elongated structures called axons. The axon extends close to the target cell that the neuron will communicate with. A neuron can send electrical signals along its axon, stimulating the release of signals called neurotransmitters, which will be received by the target cell.
• The final type of communication is physical contact, in which contact-dependent signaling molecules (which are attached to the cell surface) contact each other to send a signal.

These four groups of signaling molecules form a gigantic repertoire of possible messages in order to facilitate communication.

2. Communication is Funneled Through Receptors

How does a cell respond to a particular signal? Its ability to do this relies on the fact that a cell will only react to a signal if it has a receptor that recognizes that specific signal. Thus, a cell producing a limited number of receptors (based on its function) will restrict its responses to only those it needs.

The receptors that a cell does produce are normally displayed on its surface [4]. While displayed there, they are free to bind to a signal that they recognize [4]. This can be thought of as occurring in a fashion similar to a key (the signal) fitting into a lock (the receptor) [3]. Once this has occurred, the cell is considered to have received the signal, which will subsequently be transduced inside the cell into changes in behavior (see Figure 3).

Even with a limited set of receptors, quite complex behavior can be produced by the reception of a signal. When a single message (one signaling molecule binding to one receptor) is received by a cell many things can change—the cell could begin crawling, change direction, switch from a flat shape to a round ball, begin using up its resources, or begin creating new cell machinery. A cell possessing even a limited number of receptors is still simultaneously sensitive to all the signals that it is able to receive. Multiple signals may act together to produce responses that a single one would not generate. Animal cells rely on multiple signals to direct their behavior. This is demonstrated by the large number of signals required just for a cell to survive, The removal of these signaling molecules will cause a cell to undergo a genetic program causing its death (a process called apoptosis). Extra signals are then required on top of survival signals to produce any additional desired behaviors in the cells (for example dividing or differentiating). The large number of signals provides an animal cell with complex and subtle behavior patterns.

3. Changing a Signal into Behavior

Signal transduction is often thought of more specifically as the intracellular signaling molecules that translate a signal into changes in cell behavior.

The processes involving these molecules begin when a signal outside the cell binds to a receptor on the surface of the cell (see above). The receptor is almost always a protein that triggers steps to generate a new signal inside the cell (potentially involving many additional components) [1-4]. Intracellular signaling molecules participate in passing the new internal signal along, a process referred to as an intracellular signaling cascade (see Figure 3). This beautifully evolved mechanism involves a sequential cascade. One protein, acting as a key, will fit into the lock of a second, thereby causing the second protein to act as a key for third protein. This pattern continues until the last key turns on some internal cellular machinery that creates a response. By this method, the signaling cascade physically transfers the signal from the surface of the cell to internal machinery in other parts of the cell. During the entire process the cascade is transducing the signal into a form that is capable of interacting with this cell machinery. The entire method allows a signal located outside the cell to be interpreted internally.

In the end, it is these three components (a signal, a receptor, and a signaling cascade) that comprise the cellular nervous system, which is responsible for controlling the behavior of cells.

Additional Reading and Texts Consulted

1. Alberts et al, ed. 2002. Molecular Biology of the Cell. New York/London: Garland Publishing. 1616p.

2. Pollard TD, Earnshaw C. 2002. Cell Biology. Philadelphia: Saunders. 805p.

3. Gutkind JS. 2000. Signaling Networks and Cell Cycle Control: The Molecular basis of Cancer and Other Diseases. Totowa, NJ: Humana Press. 578p.

References

1. Gomperts et al, eds. 2002. Signal Transduction. San Diego, Calif: Academic Press. 424p.

2. Corbin JD, Francis SH, eds. 1997. Signal Transduction in Health and Disease. Philadelphia: Lippincott-Raven. 306p.

3. Frank DA, ed. 2003. Signal Transduction in Cancer. Boston: Kluwer Academic Publishers. 354p.

4. Spiegel AM, ed. 1998. G proteins, receptors and disease. Totowa, NJ: Humana Press. 324p.

(Art by Fan Sozzi)

The behavior of a cell often relies on the chemical signals it is exposed to in its environment. In general, two types of chemical signals can be found floating through the cellular environment: water-soluble (hydrophilic) molecules or fat-soluble (hydrophobic) molecules. Both are important components of cellular communication and physically interact with cellular structures to facilitate this function. In virtually every case, the cellular structures that these chemical signals interact with are receptor proteins.

Receptors and the Surface of a Cell

A cell is surrounded by a membrane, which forms a barrier between the inside and outside of the cell. This structure consists of a double layer of phospholipid molecules interspersed with cholesterol and proteins (many of which are receptors for chemical signals) that float in or are attached to the membrane. Its fat-rich composition provides it with the characteristic of being selectively permeable, allowing the flow of fat-soluble substances across it, while excluding many water-soluble ones. Fat-soluble signals, such as hormones and some vitamins, are thought to simply diffuse across the membrane. Their receptor proteins are usually found within the cell. In contrast, the vast majority of chemical signals are water-soluble and thus unable cross the fatty lipid layer of the cell membrane. Their receptor proteins must therefore span the membrane, detect their presence on the outside of the cell, and transmit this information into the interior of the cell. These proteins are called cell-surface receptors and can be thought of as a conduit for the transfer of information from the outside of the cell to the inside of the cell (see Figure 1).

Interactions Between Signals and Receptors are Highly Specific

In the media surrounding a cell, there are hundreds of chemical compounds. They are floating by, bouncing around, and rubbing against the cellular membrane. During this process, many of these compounds will make contact with cell-surface receptors, only to be sprung back into the media with no effect on the cell. The insensitivity that a cell-surface receptor shows for the mix of chemical compounds surrounding it is a result of the high specificity it has for its chemical signal. This specificity is directly related to the molecular contours of the receptor and the signal, so that they fit each other like a lock and key, becoming chemically bonded in the process. Once bonded, the receptor will transmit the presence of the signal into the cell

Classification of Cell-Surface Receptors

Although cell-surface receptors differ in the way they transmit information into the interior of the cell, in mammalian cells most can be generalized into three distinct and large families based on the mechanism they use to accomplish this transmission:

1. Ion-Channel-Linked Receptors: Converting Chemical Signals to Electrical ones

These are fast acting receptors, exemplified by the nervous system that allow sub-millisecond transmission times across synapses. Chemical signals in the form of neurotransmitters are transduced by ion-channel-linked receptors directly into an electrical signal in the form of a voltage difference across the plasma membrane. This occurs when a neurotransmitter binds to this type of receptor, altering its conformation to open or close a channel (often through or near the receptor) to the flow of Na2+, K+, Ca2+, or Cl- ions across the membrane. Driven by their electrochemical gradient (i.e. one side of the membrane has numerous ions, while the other side has few) the ions rush into or out of the cell, creating a change in the membrane potential due to the positive or negative nature of the ions. This flow of ions through the channel can trigger a nerve impulse, or alternatively stop one from occurring. (see figure 2)

2. G-protein-Linked Receptors: Clicking On Internal Switches

With hundreds of members already identified, this is the largest family of cell-surface receptors. Equally large is the diversity of chemical signals that act through G-protein-linked receptors. Some neurotransmitters that bind to ion-channel-linked receptors also bind to G-protein-linked receptors (although in a much slower fashion). Despite the diversity of signal molecules that bind them, all G-protein-linked receptors studied to date consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times. The binding of a signal to this receptor results in the switching “on” of a G-protein on the internal face of the membrane. Once activated, this G-protein will initiate a process that will alter cellular behavior. Rhodopsin, the light-activated photoreceptor in the vertebrate eye, as well as the olfactory receptors in the vertebrate nose, are members of this family. It is an ancient protein in terms of evolution, and has been found in organisms ranging from humans to yeast. There are structurally similar proteins found even in bacteria, such as bacteriorhodopsin. (see Figure 3)

3. Enzyme-Linked Receptors: Directly Activating Biochemical Activity

The study of various growth factors (extracellular signal proteins that regulate growth) shone a light on enzyme-linked receptors when it was discovered that they utilized these receptors. The responses to growth factors are typically much slower (on the order of hours) than responses connected to the other two families of cell-surface receptors. Although, recent studies have begun to connect enzyme-linked receptors to direct effects on the cytoskeleton, envolving cell movement and change of shape. Enzyme-linked receptors only span the membrane once (as opposed to seven times for G-protein-linked receptors). The internal side of the receptor acts as an enzyme, which is activated when the appropriate ligand binds to the external portion of the receptor. The largest class of receptors with this family act as tyrosine protein kinases, which phosphorylate tyrosine side chain residues on selected intracellular proteins. Such receptors are called receptor tyrosine kinases. Their function is essentially quite simple. The binding of a signal to the outer-membrane portion causes the internal kinase to switch on and begin phosphorylating cellular proteins. These phosphorylated proteins will go on to affect responses to the original signal. (see Figure 4)

Disease and Cell surface Receptors

Several human diseases have been linked to alterations in cell-surface receptors. These surface receptors activate intracellular signaling cascades. These signaling cascades regulate numerous cellular functions from cell division to cell growth and development. Defects in these proteins (usually stemming from defects in the genes that encode them) can result in disease.

The case of G-protein coupled receptors is an illustrative example. Cholera and pertussis toxins were the first to show how a GPCR could be involved in disease. In this case, the GPCR in question is the bacterial toxin that covalently modifies G protein subunits. After this discovery, many mutations in GPCRs were linked to different disorders. Defective signal of a GPCR can result from many different changes in the normal function of the receptor, such as changes in expression levels, post-translational modification, or mutations. These mutations can occur as somatic events or they can be inherited. How these mutations affect a person will depend on the origin of the mutation. In the case of a germline mutation, generalized manifestation occurs. Somatic mutations result in more focused manifestation. Some germline mutations are incompatible with normal embryonic development.

Mutations in GPCR are known to cause several disorders associated with vision. There are four visual pigments in the human eye: rhodopsin (the rod photoreceptor pigment) and the blue, green, and red cone opsins.

Reference

Spiegel AM. Defects in G-Protein-Coupled Signal Transduction in Human Disease. Annu Rev Physiol 1996; 58:143-70.

(Art by Jane Wang – note that high res versions of image files available here)

(August 2004)

Introduction

Apoptosis, or programmed cell death, is a highly regulated process that allows a cell to self-degrade in order for the body to eliminate unwanted or dysfunctional cells. During apoptosis, the genome of the cell will fracture, the cell will shrink and part of the cell will disintegrate into smaller apoptotic bodies. Unlike necrosis, where the cell dies by swelling and bursting its content in the area, which causes an inflammatory response, apoptosis is a very clean and controlled process where the content of the cell is kept strictly within the cell membrane as it is degraded [1]. The apoptotic cell will be phagocytosed by macrophages before the cell’s contents have a chance to leak into the neighbourhood [1]. Therefore, apoptosis can prevent unnecessary inflammatory response.

Apoptosis is essential to embryonic development and the maintenance of homeostasis in multicellular organisms. In humans, for example, the rate of cell growth and cell death is balanced to maintain the weight of the body. During fetal development, cell death helps sculpt body shape, separating digits and making the right neuronal connections. In the immune system, cell death eliminates B cells and T cells that elicit autoimmune response and selects the most efficient lymphocytes to encounter an antigen in the process of affinity maturation.

Pathways

Apoptosis can be triggered in a cell through either the extrinsic pathway or the intrinsic pathway. The extrinsic pathway is initiated through the stimulation of the transmembrane death receptors, such as the Fas receptors, located on the cell membrane. In contrast, the intrinsic pathway is initiated through the release of signal factors by mitochondria within the cell.

The Extrinsic Pathway: In the extrinsic pathway, signal molecules known as ligands, which are released by other cells, bind to transmembrane death receptors on the target cell to induce apoptosis. For example, the immune system’s natural killer cells possess the Fas ligand (FasL) on their surface [2]. The binding of the FasL to Fas receptors (a death receptor) on the target cell will trigger multiple receptors to aggregate together on the surface of the target cell. The aggregation of these receptors recruits an adaptor protein known as Fas-associated death domain protein (FADD) on the cytoplasmic side of the receptors. FADD, in turn, recruits caspase-8, an initiator protein, to form the death-inducing signal complex (DISC). Through the recruitment of caspase-8 to DISC, caspase-8 will be activated and it is now able to directly activate caspase-3, an effector protein, to initiate degradation of the cell. Active caspase-8 can also cleave BID protein to tBID, which acts as a signal on the membrane of mitochondria to facilitate the release of cytochrome c in the intrinsic pathway [3].

The Intrinsic Pathway: The intrinsic pathway is triggered by cellular stress, specifically mitochondrial stress caused by factors such as DNA damage and heat shock [3]. Upon receiving the stress signal, the proapoptotic proteins in the cytoplasm, BAX and BID, bind to the outer membrane of the mitochondria to signal the release of the internal content. However, the signal of BAX and BID is not enough to trigger a full release. BAK, another proapoptotic protein that resides within the mitochondria, is also needed to fully promote the release of cytochrome c and the intramembrane content from the mitochondria [4]. Following the release, cytochrome c forms a complex in the cytoplasm with adenosine triphosphate (ATP), an energy molecule, and Apaf-1, an enzyme. Following its formation, the complex will activate caspase-9, an initiator protein. In return, the activated caspase-9 works together with the complex of cytochrome c, ATP and Apaf-1 to form an apoptosome, which in turn activates caspase-3, the effector protein that initiates degradation. Besides the release of cytochrome c from the intramembrane space, the intramembrane content released also contains apoptosis inducing factor (AIF) to facilitate DNA fragmentation, and Smac/Diablo proteins to inhibit the inhibitor of apoptosis (IAP) [4].

Cancer

Cancer may arise from the dysfunction in the apoptotic pathway. Due to the sensitivity of the intrinsic pathway, tumors arise more often through the intrinsic pathway than the extrinsic pathway [5]. In the intrinsic pathway, a very common cause of tumorgenesis is mutation of the p53 protein [5]. Besides regulating apoptosis, p53 also regulates the check points in the cell cycle, DNA repair, senescence and genomic integrity [6]. Any mutation that causes p53 to lose any of its function will induce tumorgenesis by letting the cell grow indefinitely without any regulation. Another important factor in tumorgenesis is the balance between the proapoptotic and antiapoptotic members of Bcl-2 family. In a tumor cell, a mutation of Bcl-2 gene that results in increased expression will suppress the normal function of the proapoptotic proteins, BAX and BAK [5]. On the other hand, if a mutation on the BAX or BAK genes cause a downregulation of expression then the cell will also lose its ability to regulate apoptosis, again causing tumorgenesis [5].

HIV

Human immunodeficiency virus (HIV) contains many gene products that can kill the infected immune cells by activating the apoptotic pathway so that it might weaken the host’s immunity. Mainly, HIV can induce apoptosis through cell fusion (syncytia) or through its gene-encoded products. In preparation for syncytia, HIV will express one of its encoded proteins, Env, on the surface of the infected host cell. Env will bind to CD4 receptors on an uninfected cell and trigger cell fusion [7]. The fused cell will upregulate one of its cell-cycle regulating proteins, cyclin B-CDK1, which leads to the disruption of the cell cycle [7]. The disrupted cell cycle leads to more p53 production and triggers apoptosis through the intrinsic pathway by the upregulation of BAX. Besides syncytia, HIV can induce apoptosis directly through the proteins that its genes encode. For instance, Vpr can induce the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential and promoting the release of cytochrome C. Tat, on the other hand, promotes apoptosis by downregulating BCL-2 and upregulating caspase [8 7].

Conclusion

The apoptotic pathway is one of the most sophisticated pathways discovered in the cell to date. Its activity is tightly regulated and monitored by the cell. Recent advances and understanding of the apoptotic pathway have led to better and more innovative treatments against cancer and other diseases. However, the detailed mechanism of the apoptotic pathway is still waiting to be elucidated.

References

1. Raff, Martin. Cell suicide for beginners. Nature 396(1998): 119-122.

2. Csipo, I., Montel, A. H., Hobbs, J. A., Morse, P. A., and Brahmi, Z. Effect of Fas+ and Fas- target cells on the ability of NK cells to repeatedly fragment DNA and trigger lysis via the Fas lytic pathway. Apoptosis 3(1998): 105-114.

3. Adrain, C., Creagh, E. M., and Martin, S. J. Caspase Cascades in Apoptosis. Caspases-their role in cell death and cell survival. Ed. Marek Los and Henning Walczak. Moleculare Biology Intelligence Unit 24. New York: New York, 2002. 41-51.

4. Hague, A., and Paraskeva, C. Apoptosis and disease: a matter of cell fate. Nature Cell Death and Differentiation 3 September 2004: 1-7.

5. Johnstone, R. W., Ruefli, A. A., and Lowe, S. W. Apoptosis: a link between cancer genetics and chemotherapy. Cell 108(2002): 153-164.

6. Schmitt, C. A., Fridman, J. S., Yang, M., Baranow, E., Hoffman, R. M., and Lowe, S. W. Dissecting p53 turmor suppressor functios in vivo. Cancer Cell 1(2002): 289-91.

7. Gougeon, Marie-Lise. Apoptosis as an HIV strategy to escape immune attack. Nature 3(2003): 392-404.

(Art by Jen Philpott)

(August 2003)

Although seen by many as a development of the 20th century, biotechnology was, in fact, one the first technologies developed, and the earliest examples of these technologies are still the most economically important. Early use primarily consisted of methods of fermentation, both for preserving foods, and for the production of alcoholic beverages. In spite of the length of time that has passed since biotechnology came into use, it is only in the past century that we have begun to understand how these innovations work and how they can be improved. Furthermore, great strides have been made in developing new, genetically-modified microbes for these applications.

Fermentation is perhaps best described as the conversion of sugars to ethanol. It occurs via the following pathway:

During the ferment, many other byproducts of metabolism are produced. It is the sum of all of these products that gives rise to the taste and appearance of the final product.

The Essential Ingredient in Beer

The first archealogical evidence of beer production comes from ~6000 BC in ancient Babylon [1]. The discovery of how to brew beer was likely an accident, resulting from harvested barley becoming wet and being left for a time. This mix would have fermented and become a crude form of beer. It is likely that the origins of wine are similar, but with grape juice substituted for the wet barley.

Modern beer production consists of several steps, first allowing the barley to germinate [2]. This allows the starch in the barley to be degraded to sugars by enzymes in the grain. Following germination, the barley is heated to kill the sprouted plants. At this point, water and flavourings such as hops are added and the mix, or mash, is boiled to extract sugars and flavours from these ingredients. The solid particles are then filtered out of the liquid extract, or wort. Yeast is then added to the mix and is left to ferment. Although in modern beer making the strain of yeast used is pre-selected and artificially introduced, in medieval times, the yeast was carried into the mix by winds, and each beer could have several strains of yeast in it. There are still some regions where beer is produced in this manner, and this style of brewing is referred to as lambic brewing [3].

The first modern studies on beer production were conducted by the father of microbiology, Louis Pasteur. Pasteur studied fermentation, and discovered that all fermented materials contained yeast. He also found that spoiled batches of beer occurred as a result of bacterial contamination of the ferment, and this contamination could be prevented by heating the wort to 55°C before adding the yeast. This is the process of pasteurization, named after Pasteur, which is still used today. His 1876 work “Etudes sur la Biere” summarized the results of much of his work.

Today, there are two types of yeast used in beer production. The first is known as top-fermenting yeast and are used in the production of ales. Ales include a number of different styles, such as pale ales, porters, stouts, and witbiers. The other type of yeast is the bottom-fermenting yeast, used for lager production. Top fermenting yeasts are members of the species Saccharomyces cerevisiae, which are also used for the production of bread. This yeast typically ferments at relatively high temperatures (16°C-25°C). Lagers are produced by the action of Saccharomyces carlsbergensis and fermentation occurs at lower temperatures (4°C-9°C). Examples of lagers include pilsener, bock, and dunkels. There are thousands of different strains of these types of yeast used in beer production. One yeast strain used for the production of beer and wine, Saccharomyces cerevisiae, has had its genome completely sequenced. It is hoped that by using the information from the genome sequence, that better tasting beers and wines can be developed.

There are 15 different yeast genera that are generally accepted as a part of wine-making (oenology), and include the following species: Brettanomyces and its sexual equivalent Dekkera; Candida; Cryptococcus; Debaryomyces; Hanseniaspora and its asexual counterpart Kloeckera; Kluyveromyces; Metschnikowia; Pichia; Rhodotorula; Saccharomyces; Saccharomycodes; Schizosaccharomyces; and Zygosaccharomyces [4]. Of the yeast found on the grapes themselves, Kloeckera and Hanseniaspora make up 50-75% of the total yeast population. Industrial winemaking requires careful selection of the yeast used in fermentation, as each yeast strain imparts certain flavour characteristics on the final product.

Depending on whether the wine being produced is white or red, the skins will be left in the juice during the fermentation or will be removed. The red colour of red wine comes about primarily from the skin. If cultured yeast (guided fermentation) are going to be used during the fermentation, they will be added at this point. The cultures used for inoculation are primarily Saccharomyces sp., similar to those used in beer production.

On Using Saccharomyces…

The reasons for the use of Saccharomyces in beer and wine production are relatively straightforward. Both beer mash and grape juice are mixtures characterized by a high concentration of sugar and low pH. During fermentation these sugars lead to the production of ethanol and carbon dioxide. It is important that the yeast strain used is able to survive the higher ethanol concentrations produced. For beer, these concentrations generally range from 3-9%, while for wine they are substantially higher at 11-15%. Saccharomyces strains are fairly resistant to high ethanol concentrations. Even when present as a very small percentage of the initial yeast population, these strains tend to be the dominant strain at the end of natural fermentation due to their ethanol tolerance [4].

Saccharomyces strains are also chosen based on their ability tolerate other compounds, such as sulphite [4]. In modern winemaking, the native yeast strains are not killed by heat as they are in beer-making, because the act of heating alters the final taste of the wine. Instead, the grape juice is treated with sulphite to remove the native strains, thus making it necessary for the Saccharomyces strains to survive the addition of these compounds.

In addition to these practical reasons for choosing Saccharomyces strains, vintners may also want to impart a particular characteristic to the final wine produced. For this, the winemaker may select strains that have been characterized in the past as having a particular taste. Thus, there is a great deal of trial and error in selecting yeast strains. It is also not yet entirely clear what genes are responsible for giving a particular yeast strain a particular flavour in the final product. A partial list of desired characteristics in wine is given in the table below.

Wine Production Can Mean Twice the Fermentation

Following the primary fermentation of wines, several secondary fermentations may be carried out, depending on the final product desired. During the production of many wines, two main acidic components are present: malic and tartaric acid. The final concentration of these organic acids determines the final acidity of the wine produced [4]. Quality wines may undergo a secondary fermentation, known as malolactic conversion, to decrease the acidity of a wine. In this process, a lactic acid bacterium known as Oenococcus oeni is introduced into the grape juice and catalyzes the conversion of malic acid to the less acidic-tasting lactic acid [4]. This conversion is important not only because it improves the palatability of a wine, but it can increase shelf-life as well. One problem with this process is that this secondary fermentation often fails to progress very quickly. This leads to increased spoilage products in the wine, and and in particular produces biogenic amines, the main culprit behind wine-induced headache [5].

Several non-desirable metabolites can be produced during wine fermentation, such as biogenic amines, as mentioned above. Biogenic amines, which are byproducts from the metabolism of amino acids [5], are vasodilators, and therefore can induce headaches in approximately one in three wine-drinkers. Urea is also produced during the fermentation. Excess urea in wine can lead to the production of ethyl carbamate, a known carcinogen. Research being conducted at UBC is currently working on these problems by developing yeast strains that can carry out the malo-lactic conversion, without the need for a second, inconsistent fermentation and strains that are capable of degrading urea to harmless compounds [6, 7].

Different types of secondary fermentations are also carried out in the production of sparkling white wines and sherry. For champagne, this consists primarily of adding fermentable sugar to the bottle when the wine is added. Following corking, fermentation takes place in the bottle, producing more ethanol and carbon dioxide. The carbon dioxide dissolves into the wine, producing the carbonation observed when uncorked. Sherry involves an interesting process, whereby a stack of barrels, known as a solera, are used. The wine base (product of the primary fermentation) is added to the top barrel and allowed to age for a period of several months. Then half of the wine from the top barrel is added to the wine in the barrel below it. This is continued every few months until the wine in the bottom barrel is ready for bottling. This process permits greater oxygenation of the wine. Higher oxygenation is required with sherry wines to promote the growth of a flor, or film, of yeast on the barrels and on the surface of the wine [8]. The yeast strains involved forming the flor are Torulaspora fermentati, Zygosaccharomyces rouxii, and Saccharomyces cerevisiae. This combination of yeasts produces acetaldehyde and glycerol at higher concentrations than in the primary ferment, resulting in the distinct flavour of sherry. At the end of the entire process, the wine may have spent up to 10 years in a solera.

Wine and beer are both used in the production of another important food product: vinegar. This process requires that a vinegar generator first be created (Figure 2). This consists of a large wooden vat, approximately 8 metres in height and 4 metres in diameter, which is loosely packed with wood shavings. The shavings act as a surface on which the bacteria can grow. An alcohol source is then poured over the wood shavings; beer is used if malt vinegar is desired, while wine is used to produce wine vinegar. Following its first run through the reactor, the liquid is collected and run through the generator several more times. When the acetic acid content rises above 4%, it can be collected and bottled as vinegar [2]. The oxidation of ethanol to acetic acid is carried out by Acetobacter sp. For the production of purified acetic acid or white vinegar, the acetic acid solution produced by this reactor is collected and distilled. The pure acetic acid is then diluted to the desired concentration to be sold as white vinegar. High-quality vinegars may be allowed to age, during which time esters form from the reaction of acetic acid and residual ethanol. This allows the formation of very complex mixtures of flavours that are not found in the beer or wine alone.

Despite the fact that these technologies have been with humanity for millennia, they are still among the most economically important products of biotechnology. Annual beer production is now over 1 billion barrels, while for wine it is over 600 million barrels. These represent billions of dollars of sales and tens of thousands of jobs. Research is seeking to make the products of beer and wine production even better and safer than they have ever been before.

References

1. Brown, C.M., Campbell, I. and F.G. Priest. 1987. Introduction to Biotechnology. Blackwell Scientific Publications, Oxford.

2. Brock, T.D., Madigan, M.T., Martinko, J., and J. Parker. 1994. Biology of Microorganisms. Prentice Hall, New Jersey.

3. http://www.beer-brewing.com

4. Pretorius, I. 2000. Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast. 16:675-729.

5. Lonvaud-Funel, A. 2001. Biogenic amines in wine: the role of lactic acid bacteria. FEMS Microbiology Letters. 199:9-13.

6. http://www.agsci.ubc.ca/wine/research.htm

7. Volschenk, H., Viljoen-Bloom, M., Subden, R.E., and H.J. van Vuuren. 2001. Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae. Yeast. 18:963-970.

8. Sandeman, G.G. Sons & Co. 1955. Port and sherry: the story of two fine wines. George G. Sandeman Sons, London.

(Art by Jane Wang – note that high res versions of image files available here)

Advocates of biotechnology have consistently upheld the notion that the field contributes to human health care, environmental protection, and industrial-scale synthesis of complex chemicals. Despite the obvious importance of these areas, biotechnology also has much to offer to other fields. One of the oldest of these is the mining industry. The earliest use of microbial processes for mining occurred long before it was clear that microbes were responsible for the effects observed. At the Rio Tinto (Red River) mine in Seville, Spain, Roman era copper mine workings were rediscovered in 1556. Evidence suggests that the mine used water from the Rio Tinto – water containing a very high concentration of ferric iron owing to microbial activity in the area1. When the water from this river was irrigated onto copper containing deposits, the copper dissolved and later precipitated as smaller deposits that could be collected. Although the people at the time likely believed this process to be magic, we now know that it was the first recorded use of biomineralization.

Bacteria hard at work: Biomineralization

A number of genera of bacteria have been shown to be important in biomineralization, including: Acidothiobacillus (previously Thiobacillus), Leptospirillum, Acidiphilium, Sulfobacillus, Ferroplasma, Sulfolobus, Metallosphaera, and Acidianus. Interestingly, these microbes represent Gram-negative, Gram-positive bacteria and Archaea, indicating that even within a superficially simple and inhospitable environment like a mine tailings pond, a remarkable diversity of life can be observed.

Bacteria involved in biomineralization are usually highly acid-tolerant, mesophilic (20-40°C temperature optimum) or thermophilic (40-70°C temperature optimum) chemolithoautotrophs. They are usually able to use reduced sulphur compounds and/or ferric (Fe²⁺) iron as electron sources. In most ore deposits both of these molecules are readily available in the form of iron pyrite (FeS₂). Following exposure of buried pyrite to water and oxygen, a spontaneous oxidation takes place according to the following reaction:

This reaction begins to produce ferrous iron (Fe³⁺) – a powerful oxidant that is further capable of oxidizing mineral sulphides:

The most important product of these reactions is sulfuric acid (H₂SO₄). As a result, these reactions begin to alter the pH of the environment to favour the growth of acid-tolerant microbes that are important for biomineralization. The most common organism found is Acidothiobacillus ferroxidans (previously known as Thiobacillus ferroxidans). Once this bacteria begins to grow it uses the Fe²⁺ released in the second reaction as an electron source, thus regenerating Fe³⁺, the ion capable of oxidizing even more of the mineral sulphides. This process will continue until the pH of the water in the pile approaches 1 [1]. The entire process described above is known as acid mine drainage, and can be very environmentally destructive. The acid mine runoff is highly acidic and contains dissolved iron concentrations. If the runoff from the mining operation enters the surrounding environment, it can render entire streams, rivers, and lakes completely devoid of any eukaryotic life.

Consequently, when there is a risk of acid mine drainage in traditional mining operations, special care must be taken to prevent it. These preventative measures can include a number of techniques. These are designed to prevent either the initial oxidation from occurring, or the subsequent acidification of the water. Acidification can be interrupted by combining the sulphide-containing ores with material, such as limestone, that is capable of neutralizing the acid produced by the oxidation. This prevents the positive feedback loop generated by the bacteria.

How to Extract Ore Without Anaesthetic: Bioleaching

Despite the remarkably destructive potential of acid mine drainage, there is a silver lining to this industrial dark cloud. Many minerals are capable of forming sulphidic deposits, including copper, uranium, nickel, and cobalt. If large quantities of these metal sulphides are found, they can be commercially extracted using the principle of acid mine drainage. There are two approaches to tackling this type of operation. The first is called irrigation style bioleaching, one example of in situ bioleaching (Figure 1). In order to be considered feasible, the deposit itself must be relatively permeable to water, while the entire site is contained in geological structures that are impermeable to water, so that the mine drainage cannot enter any waterways. Artificial piles can also be created from crushed low-grade ore that is piled onto impermeable liners up to heights of 350 m.

Regardless of whether the pile is artificial or carried out in situ, sulfuric acid, water, and air are then pumped into the deposit over a period of weeks to months. The reactions involved are similar to those above, except that the metal sulphide partially replaces the FeS2 represented. Following an incubation period, the liquid is then removed by pumping. This runoff is very acidic, and contains a high concentration of the metal of interest. The metal can then be isolated by a number of conventional techniques, including precipitation or electroplating. Approximately 20% of the world’s copper is produced by bioleaching, and that number is expected to rise in the years ahead [2, 3]. Within Canada, this type of process has been used to extract uranium from the Elliott Lake district in northern Ontario. For in situ piles, fresh water and an acid neutralizer can then be pumped into the deposit until standards within the runoff are below a minimally acceptable threshold.

Special Treatment for Special Ores: Stirred Tank Processes

The other major form of bioleaching is known as stirred tank processes. The name is fairly self-explanatory, as the process requires constructing large aerated tanks that are generally arranged in a series, so that runoff from one tank serves as raw material for the next (Figure 2). In this way, the reactor can operate in continuous flow mode, with fresh ore being added to the first tank while the runoff from the final tank is removed and treated. The ore to be processed is generally crushed to a very small particle size, to ensure that the solids remain suspended in the liquid medium. Mineral nutrients in the form of (NH₄)2SO₄ and KH₂PO₄ are also added to the tanks to ensure maximal microbial density is maintained.

Due to the extremely high cost of stirred tank reactors, they are only used for highly valuable materials. For gold extraction for example, this technique is usually used when the ore body contains high concentrations of arsenopyrite (AsFeS). This compound is extremely recalcitrant to chemical reactions and effectively shields the gold from the cyanide that is used to dissolve it. Thus, unlike irrigation-style bioleaching, where the liquid contains the material of interest, stirred tank processes are used as a pretreatment to remove low value sulphide material from higher value minerals, such as gold. The solid particles leaving the last aeration tank are then moved to a facility for chemical processing. This pretreatment can increase the yield of gold-bearing ore by twenty times over traditional crushing and immediate chemical extraction. These reactors can be truly enormous, with one such facility consisting of a series of 24 tanks, each holding 1 000 000 litres of crushed ore and liquid [4].

Even Bacteria Have to Deal with Waste Management…

Yet another example of biotechnology in mining involves a process called metal precipitation. This process is used to treat wastewater from mining operations, which, as described above, contains high concentrations of dissolved SO₄^2- and Fe²⁺, as well as excess acid. This material cannot be released directly into the environment as it is extremely hazardous to stream and river ecology. In what is essentially a reversal of the procedure described that leads to acid mine drainage, sulphate-reducing bacteria like Desulfovibrio and Desulfotomaculum oxidize organic matter or H₂ by using sulphate as an electron acceptor according to the following reactions:

The sulphide present then immediately reacts with any dissolved metal, producing an insoluble metal sulphide:

The metal sulphide is very inert and can be disposed of, provided it is not exposed to oxygen, lest the process begin again.

In many mining operations, this process is carried out in engineered anaerobic swamps. A large enclosure is first lined to make it impermeable. The wastewater is then allowed to enter the area where the pH is adjusted with limestone. As the pH increases, some iron precipitates and the rest of the material is passed into an area containing organic waste material. The degradation of the organic waste maintains the anoxic state of the containment pond. Microbial action reduces the sulphate present to metal sulphides that are then permanently precipitated within the passive reactor. The top of the reactor can be covered with topsoil and planted to restore the aesthetic potential of the area.

Biotechnology has played a pivotal role in the mining industry for hundreds of years. The use of bacteria has enabled for more efficient ore extraction, and purification of ore compounds. In more recent years, research has been devoted to developing more environmentally sound and efficient extraction processes and microbial remediation of mining sites. Future developments will likely include increasing the temperature at which stirred tank reactions can be carried out, by identifying or developing bacterial strains that have higher optimal temperatures. Higher temperatures translate into elevated reaction rates and evading the necessary cooling of these systems – an extremely energy intensive step. In addition, bacteria that are better able to resist the abrasion from the stirred tank process will also be very useful. These initiatives may also be much less environmentally destructive than conventional technologies – what we can hope to be the beginnings of a new generation of mining technologies.

References

1) Davis Jr., R.A., Welty, A.T., Borrego, J., Morales, J.A., Pendon, J.G. and J.G. Ryan. 2000. Rio Tinto estuary (Spain): 5000 years of pollution. Environmental Geology. 39: 1107-1116.

2) Brierley, C.L. and J.A. Brierley. 1997. Microbiology for the Metal Mining Industry. in Manual of Environmental Microbiology. (Ed.) C.J. Hurst. ASM Press, Washington D.C.

3) Brierley, C.L. 1995. Bacterial oxidation. Engineering and Mining Journal. 196:42-44.

4) Acevedo, F. 2000. The use of reactors in biomining processes. Electronic Journal of Biotechnology. 3: online.

Additional Reading

Rawlings, D.E. 2002. Heavy metal mining using microbes. Annual Review of Microbiology. 56:65-91.

(Art by Jane Wang – note that high res versions of image files available here)

(August 2004)

Animal or plant cells, removed from tissues, will continue to grow if supplied with the appropriate nutrients and conditions. When carried out in a laboratory, the process is called Cell Culture. It occurs in vitro (’in glass’) as opposed to in vivo (’in life’). The culture process allows single cells to act as independent units, much like a microorganism such as a bacterium or fungus. The cells are capable of dividing, they increase in size and, in a batch culture, can continue to grow until limited by some culture variable such as nutrient depletion.

Cultures normally contain cells of one type although mixed cultures, especially of bacteria, are common in food sciences and wastewater treatment related studies. The cells in culture may be genetically identical (homogenous population) or may show some genetic variation (heterogeneous population). A homogenous population of cells derived from a single parental cell is called a clone. Therefore all cells within a clonal population are genetically identical.

Why Grow Cells in Culture?

There are a number of applications for animal cell cultures:

• To investigate the normal physiology or biochemistry of cells. For instance studies of cell metabolism.
• To test the effect of various chemical compounds or drugs on specific cell types (normal or cancerous cells, for example).
• To study the sequential or parallel combination of various cell types to generate artificial tissues e.g. artificial skin. Possibility of generating artificial tissues is an emerging and intensively studied area of biotechnology known as “tissue engineering”.
• To synthesize valuable biologicals from large scale cell cultures. The biologicals encompass a broad range of cell products and include specific proteins or viruses that require animal cells for propagation. For example, therapeutic proteins can be synthesized in large quantities by growing genetically engineered cells in large-scale cultures. The number of such commercially valuable biologicals has increased rapidly over the last decade and has led to the present widespread interest in animal cell culture technology.

The major advantage of using cell culture for any of the above applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells. The disadvantage is that, after a period of continuous growth, cell characteristics can change and may become quite different from those found in the starting population. Cells can also adapt to different culture environments (e.g. different nutrients, temperatures, salt concentrations etc.) by varying the activities of their enzymes.

Milestones of Cell Culture

The history of cell culture dates back to early twentieth century. The original impetus for the development of cell culture was to study, under the microscope, normal physiological events such as nerve development. The growth rate of animal cells is relatively slow compared with bacteria. Whereas bacteria can double every 30 minutes or so, animal cells require anywhere from 18 to 24 hr to double. This makes the animal culture vulnerable to contamination, as a small number of bacteria would soon outgrow a larger population of animal cells. Consequently, animal cell culture did not become a routine laboratory technique until the 1950s.

The need for cell culture, especially at large scales, became apparent with the need for viral vaccines. Major epidemics of polio in the 1940s and 1950s promoted a lot of effort to develop an effective vaccine. When it was shown in 1949 that poliovirus could be grown in cultures of human cells, considerable interest was shown to develop large quantities of the polio vaccine using cell culture. The polio vaccine, produced from de-activated virus, became one of the first commercial products of cultured animal cells.

Recombinant DNA technology (also known as genetic engineering) was developed in the 1970s to express mammalian genes in bacteria. It soon became apparent that large complex proteins (and especially those having therapeutic value) couldn’t be produced in bacteria, as they do not have the appropriate metabolism to add sugar chains to these proteins. Therefore, genetically engineered animal cells were developed for large-scale commercial production of such important proteins.

Another milestone in the animal cell culture technology came in 1975 with the production of hybrid cells (known as hybridoma) from the fusion of two or more cells capable of continuous production of a single type of antibody. These antibodies have diagnostic and therapeutic value and are now produced commercially in kilogram quantities from large-scale cultures of hybridomas.

Plant Cell Culture

Plant cells have been cultured to produce many ingredients needed by the food industries. Tremendous progress has been made in recent years in understanding the basics of plant metabolism and in the development of bioprocesses as well as design and operation of large-scale bioreactors for plant cell culture. A wide range of food ingredients including flavors, colorants, essential oils, sweeteners and antioxidants have been produced in culture. Japan has so far been the most successful country in the world to carry out plant cell culture on commercial scale. Ginseng products derived from cell suspension cultures of Panax ginseng used as an additive in wine, tonic drinks and herbal liquors have been produced by a company in Japan since 1990 with a net sale of 3 million dollars in 1995.

Types of Mammalian Cultures

Freshly isolated cultures from mammalian tissues are known as primary cultures until sub-cultured. At this stage, cells are usually heterogeneous but still closely represent the parent cell types as well as in the expression of tissue specific properties. After several sub-cultures onto fresh media, the cell line will either die out or ‘transform’ to become a continuous cell line. Such cell lines show many alterations from the primary cultures including change in morphology, chromosomal variation and increase in capacity to give rise to tumors in hosts with weak immune systems.

Animal cells can be grown either in an unattached suspension culture or attached to a solid surface. Suspension cultures have been successfully developed to quite large bioreactor volumes, with successful production of viruses and therapeutic proteins.

The range of commercially available biologicals produced by cell culture technologies has increased rapidly over the last decade. This is particularly so for therapeutic proteins synthesized from selected or genetically engineered mammalian cells. They are needed in large quantities and hence require careful study of the underling biochemical, cell biological and engineering principles for control of production processes.

(August 2004)

To the scientific community, biotechnology is a field of study aimed at understanding how things work at the cellular or biochemical level; with the ultimate goal of deploying that knowledge to control or manipulate life. One topic in biotechnology that swings between the practical and the conceptual is the field of cryobiology.

Mike Myers in Austin Powers: The Spy who Shagged Me, Tom Cruise in Vanilla Sky and Mel Gibson in Forever Young, have all introduced us to the idea of “cryonics.” In fact, the concept of freezing and reanimating organisms is not only accepted but believed to be real. The popularity and actual existence of cryogenic companies add to the confusion on whether this technology actually exists and works. If you follow their marketing, cryogenic companies will eagerly collect your money to give you a chance at their “life-extending” possibilities. But will this be just a very frigid burial? Before you sign over your life savings, read this article and learn what applications of cryonics are practical at this time.

Defrosting a Mystery: Cryobiology Defined

Cryobiology comes from three Greek words: “kryos” meaning cold, “bios” meaning life and “logos” meaning discourse of study. Cryobiology is the science of studying the effects of very low temperature on life. Living organisms all share basic building blocks. These blocks include the genetic library held by the DNA double helix that is transferred to RNA in order for macromolecules like proteins, polysaccharides and lipids, to be manufactured. Despite their importance it is H₂0, aka water, that is the most fundamental building block to all life. Water is the key to life because it is the primary solvent in all living creatures. In cryobiology the nature of water’s transformation from a liquid solvent to a solid structure during freezing provides for the ability to preserve or destroy. Understanding how to manipulate the freezing process is the cryobiologist’s ultimate goal.

Through evolution, certain organisms have adapted to survive at low temperatures well below the freezing point of water (0°C). These organisms have the ability to create biomolecules that act as anti-freeze, lowering the temperature at which intracellular and to some extent, extracellular ice forms. In Figure 1, the scale of standard physiological temperatures for several species is shown. At -20°C, the Himalayan midge is still biologically active and is therefore able to prevent water from freezing in its cells.

Just as food is stored in the deep freezer, low temperatures have the ability to preserve life. Intact DNA and proteins from the woolly mammoth can be still be found some 50,000 years later in the frozen tundra of Siberia. On the other hand, these temperatures also have the ability to destroy. Frozen crops or frostbite on ears or toes are examples where the cold has damaged cells so badly that they are destroyed. It is this dual nature of the cold to preserve and destroy that has created its own special field of cryobiology in the entire subject of biotechnology.

Out of the Ice Age: History of Cryobiology

Cryobiology may sound like a brand new field created in the 21st century, but in fact the first documented study can be traced back to Sir Robert Boyle in his 1683 monograph “New Experiments and Observations Touching Cold”, in which this famous physicist documented the effects of freezing on living animals. Further research was continued through the 18th and 19th centuries, but it was not until the mid 1900’s that real progress was reported. In the 1940s, scientist Chris Polge of the University of Cambridge, accidentally discovered the cryoprotective abilities of glycerol through the mislabeling of reagent bottles. Using glycerol, the scientist Peter Mazur in 1963 conducted experiments to model the mechanisms of freezing within cells. These early studies on the cold have underscored the recent developments in cryobiology today.

Frozen Solid: Challenges and Obstacles in Cryobiology

From the fundamental research on the modeling of cells during the freezing process conducted by the scientist Mazur, it was discovered that it is the rate of cooling that determines the survival rate and type of damage that cells experience. To prevent irreparable damage, cells must not be cooled too slowly, or too quickly.

When cells are cooled too slowly, the outside environment of the cell freezes first and extracelluar ice forms. Extracellular ice creates a chemical potential difference across the membrane of cells creating osmosis of water which dehydrates and shrinks the cell. The slower the cells are cooled, the longer cells are dehydrated causing irreparable damage termed “solution injury” (Figure 2B). On the other hand, when cells are cooled too quickly, the cell retains water within the cell. The water expands when frozen and intracellular ice damage forms (Figure 2F). The abrasive ice crystals physically destroy the cell itself, termed “intracellular ice injury”. Both mechanisms, if balanced perfectly would achieve a maximum survival rate where the total cell damage from both mechanisms is minimized (Figure 2D). Specific cell properties such as membrane permeability to water and initial intracellular water concentration will determine the precise rate of cooling as shown in Figure 3.

Fun with Refrigeration: Current Topics and Research in Cryobiology

Understanding how the cold works on the cellular level is the basis of several specialized cryobiology focus areas: cryosurgery, cryopreservation and cryonics (as known as cyrobiosis).

In cryosurgery, the power of the cold to destroy cells, rather than preserve, is used to surgically operate on cells. Low temperature exposure (usually -20°C) can kill undesirable cells like cancer cells. In addition to the two types of cellular damage discussed earlier (solution injury and intracellular ice injury), exposure to the cold can eliminate blood circulation in the undesirable cells, thereby preventing the delivery of nutrients and oxygen as well as the removal of wastes and CO2. Finally, when the body thaws, the body’s immune system naturally detects and quickly removes the damaged cells.

In cryopreservation, the power of the cold to preserve cells is used to store or keep desirable cells in a state of suspended animation. Cell and tissue banks of frozen cells help scientists manufacture cellular products and conduct experiments on cells. So far only thin tissue cells like skin cells can be successfully frozen and unthawed. Extending this success to larger tissues and organs is a challenge in future cryopreservation research.

In cyronics or cryobiosis, the power of the cold is harnessed to preserve whole organisms, beyond just cells and tissue. This is the most well-known application of cryopreservation, but also the most controversial and so far unproven. Despite the scientific shortfalls such as the lack of a revival method, there are believers in this technology and have signed up as participants for their own preservation. In April 22, 2001, an article from the New York Times Magazine reported that in the United States about 90 people are “suspended” nationwide and the number continues to increase as more and more believers are frozen post-mortem.

Putting it on Ice: Conclusion

Both cryosurgery and cryopreservation are technologies with promising success given our understanding of the science of cryprotectants and freeze-thaw protocols while cryonics remains a meeting of the “high-tech” and the “sci-fi” which should be dreamed rather than tried. However cryonic service companies promote promises of life extension past the current technological limitations in hopes of a future remedy for our current level of technology. It is a risky proposition to invest in a conceptually appealing technology that lacks scientific validation. The impracticality of these scientific claims may be only dispelled over time with further innovative research.

(Art by Jiang Long)

(August 2004)

Generally, this term refers to food crops that have been altered using a variety of molecular biology techniques in order to provide them with either new or enhanced characteristics. Examples of such enhancements of modifications are herbicide tolerance, pesticide resistance, greater nutritional content or increased tolerance of cold temperatures. Genetically modified organisms (GMOs) can also be referred to as transgenic organisms. Transgenic simply means that the organism’s genes come from more than one source.

The idea of enhancing desired traits in food crops is not new. Upon domestication of many plants, farmers used the process of artificial selection to grow plants with desired qualities. However this method can be time consuming and it is very difficult to introduce new traits into a specific population. In contrast, using genetic engineering, scientists can take the gene that controls the trait from one organism and insert it into another organism that does not have the gene. This creates an organism with the desired characteristic quickly and easily. A common example of genetic engineering is the insertion of Bacillus thuringiensis genes into corn to make Bt corn. Bacillus thuringiensis is a bacterium that naturally produces a protein that is lethal to insect larvae. By transferring the genes that encode this protein into corn, scientists have created a type of corn that produces its own pesticides, making it resistant to insects such as the European corn borer.

Transferring the gene

Taking a gene from one organism and inserting it into another is essentially a process of cutting the gene which codes for the trait of interest from the foreign organism and pasting this gene into the genome of the organism that you want to alter.

Let us use the insertion of B. thuringiensis genes into corn as an example. In order to cut out the gene of interest in the bacteria, its total DNA is isolated. Special enzymes, called restriction endonucleases, act as scissors to cut out the desired gene. These enzymes are sensitive to the DNA sequence and will only cut DNA at specific spots. There are many different enzymes that cut in different places, so the enzyme used depends on the sequence of DNA surrounding the desired gene.

Once the gene is cut out, scientists must make an “expression cassette.” This consists of additional DNA surrounding the gene so that the corn cell knows where the gene of interest begins and ends. The part that tells the corn cell where the gene begins is called the promoter and the end, the terminator. Once the expression cassette has been made, it is inserted into a plasmid. The plasmid is a parasitic circle of DNA present in bacteria. By putting the cassette into a plasmid, millions of copies of it can be made. These copies are then introduced into the host cell and get inserted into the genome. Cells which have successfully incorporated the foreign gene into their genome are then expanded in cell culture and used to generate new plants.

The ethics of GM Foods

GM foods have been the subject of much controversy. Advocates feel that GM foods will help provide food to the world’s continually expanding population. Since the number of people on earth keeps increasing (over 6 billion, and expected to double within 50 years), and the amount of land suitable for farming remains constant, more food must be grown in the same amount of space. Genetic engineering can make plants that will give farmers better yields through several different methods.

Crops can be harmed or destroyed by many different factors. Insects, weeds, disease, cold temperatures and drought can all adversely affect plants resulting in lower yields for the farmer. Genetic engineering techniques can be used to introduce genes, creating plants that are resistant or tolerant to these factors. Bt corn is an example of the introduction of a pest resistance gene. Monsanto has created strains of soybeans, corn, canola and cotton that are resistant to the weed-killer Roundup®. The weed-killer can be sprayed over the entire crop, killing all plants except the transgenic crop intended to be grown. Scientists have also taken a gene from a cold-water fish and introduced it into potatoes to protect the seedlings against sudden frost. These methods all create plants that are more likely to survive and be healthy, thereby increasing the production of farmer’s fields.

Genetic modification can also be used to change the properties of the crop, adding nutrients, making them taste better, or reducing the growing time. A good example of adding nutrients to food is the development of “golden” rice. Many countries in the world rely on rice as their primary food source. Unfortunately, rice is missing many essential vitamins and minerals, so people whose diet is based on rice are often malnourished. One of the most severe consequences of this is blindness caused by vitamin A deficiency. Researchers at the Swiss Federal Institute of Technology Institute for Plant Sciences genetically engineered rice, making it high in vitamin A. The group hoped to distribute the rice for free to any third world country requesting it.

Golden rice is a controversial subject in its own right. Its development was a breakthrough for biotechnology as it was the first time 3 genes were introduced simultaneously (generally, only one gene is transferred at a time). Mammals make vitamin A from beta-carotene, which is not found in polished white rice. A precursor to beta-carotene (geranyl geranyl diphosphate, or GGPP) is present, but three additional chemical reactions must be carried out to transform GGPP into beta-carotene. The gene transfer was successful, resulting in rice that is high in beta-carotene and is actually yellow coloured. On the surface, this seems like the solution to vitamin A deficiency.

Sounds great, right? GM foods can be grown easily, withstanding cold or drought, without spraying for pests or weeds. Not only that, but the food can be made more nutritious. So what’s the problem? Why so much controversy?

Opponents of genetic modification have many criticisms against this new technology. First of all there are multiple environmental concerns. GM foods can cause harm to other organisms unintentionally. For example, a study published in Nature on Bt corn found that the pollen caused high mortality rates in monarch butterfly caterpillars, even though the caterpillars don’t eat corn [1]. If the Bt corn pollen is blown onto neighbouring milkweed plants (the caterpillars food source) the caterpillars could eat the pollen and die. The results of this study are under debate, since the experiments were not done in the field, but in a laboratory, and new studies suggest that the original may be flawed. Researchers at the University of Guelph performed a study and found that under natural conditions, Bt corn does not pose a risk to the monarch butterfly [2].

Similarly, if pollen is blown onto neighbouring plants, the plants could crossbreed and the introduced gene could be transferred to non-target plants. This is a concern if a herbicide resistant crop were to breed with a weed and transfer the herbicide resistance gene. This would create a weed that is unharmed by the chemicals used to kill it.

Monsanto has patented their Roundup Ready seeds, and farmers wishing to use them must purchase a license from the company. This can lead to trouble for farmers who don’t use the Monsanto seeds. Perry Schmeiser is a canola farmer in western Canada who has never bought seeds from Monsanto. In 1998 he was sued by Monsanto since they discovered Roundup Ready canola in his field. Schmeiser claims that the seed was blown in from neighbouring fields, but Monsanto believes he obtained it illegally or stole it. Regardless of how it was obtained, Monsanto felt this was patent infringement and took Schmeiser to court in June of 2000. This court battle captured the interest of farmers around the world, because even if they did not intend or even want to have patented seeds in their fields, they could be sued. The judge ruled in favour of Monsanto and stated that it didn’t matter how the seed got into Schmeiser’s field. Whether it was blown in, cross-pollinated by birds, bees or animals, fell off farmer’s trucks or migrated from a neighbour’s field, it is still patent infringement, and the plants were to become the property of Monsanto. All of Schmeiser’s profits from 1998 were awarded to Monsanto since there was a probability of having the genetically altered seeds throughout his fields.

Insect pests may also become resistant to the toxins produced by GM crops like Bt corn. It is now known that some bacteria are becoming antibiotic resistant (so-called “superbugs”) making it difficult to treat diseases such as tuberculosis. Likewise, opponents of GMOs believe that insects could become pesticide resistant making them difficult to control in the future.

Along with environmental concerns, there are also worries about the effects that GM foods can have on humans. There are concerns that introducing a new gene into a food could cause an allergic reaction in some people (for example, if the gene came from a nut). Most scientists believe that other than allergic reactions, GM foods do not pose a threat to human health, however as with all new products, no long-term studies have been performed.

How are genetically modified foods regulated in Canada?

The Canadian Food Inspection Agency (CFIA) is responsible for the control of GM foods in Canada. The CFIA has strict criteria that must be met before a GM food can be marketed. These include: how the food crop was developed, including the molecular biological data which characterizes the genetic change; composition of the novel food compared to non-modified counterpart foods; nutritional information for the novel food compared to non-modified counterparts; potential for new toxins; and potential for causing an allergic reaction. Once the government is satisfied that requirements have been met, the food is approved for consumers. Right now, Canada has no mandatory labeling policy for GM foods, it is strictly on a volunteer basis, however mandatory labeling is required if the introduced gene poses an allergy risk (eg. if the gene introduced came from a nut) or if the food’s nutritional content has changed. A Canadian standard for labeling of biotechnology derived foods is being developed and is expected to be completed in the fall of 2002.

The CFIA has approved 51 “novel foods”, most of which are GM foods, including corn, (types resistant to corn borers and herbicides); canola, (varieties resistant to herbicides); potato (varieties resistant to Colorado potato beetles); tomato (varieties that ripen slowly); squash; soybean; sugarbeet; flax; and cottonseed oil.

References

1. Losey JE, Rayor LS, Carter ME. Transgenic pollen harms monarch larvae. Nature 399, 214 (1999).

2. Sears MK, Hellmich RL, Stanley-Horn DE, Oberhauser KS, Pleasants JM, Mattila HR, Siegfried BD, Dively GP. Impact of Bt corn pollen on monarch butterfly populations: A risk assessment. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):11937-42.

(Art by Jen Philpott)