Metro State Atheists

Promoting Science, Reason, and Secular Values

Why Christians can’t disregard The Gospel of Thomas

In a recent debate with a Christian, he told me that The Gospel of Thomas is “simply a heretical forgery, much the same as the Gospel of Judas, the Gospel of Mary, and the Gospel of Philip.” After an extensive amount of research, I have found that his statement has some profound ramifications. I went through all 114 sayings in The Gospel of Thomas and found that approx. 84%* of the sayings are contained within the Canonical Gospels, either verbatim or slightly changed in wording. Some of these sayings are the “important” ones. (Examples of this are below1) Ones that most Christians themselves point to about how compassionate Jesus was, such as “turn the other cheek” and “love thy neighbor as thyself”, are in the Gospel of Thomas. (Sources and characters are at the bottom)

When was the Gospel of Thomas written?

Experts, through radiometric dating, have dated the Gospel of Thomas found at Nag Hammadi to being written between 50-140CE. This is startling, considering the Gospel of Mark, at its earliest, was written between 65-80CE. At the earliest, that is at least 15 years after the Gospel of Thomas was written. One then should be able to conclude then, that writer of The Gospel of Mark simply took the sayings from The Gospel of Thomas and inserted them into stories. He plagiarized the Gospel of Thomas, to use today’s legalistic terms.

What does this all mean?

This means that one cannot make the statement “[The Gospel of Thomas] is simply a heretical forgery, much the same as the Gospel of Judas, the Gospel of Mary, and the Gospel of Philip.” and yet continuously quote scripture derived directly from it. If the Gospel of Thomas is nothing more than a “heretical forgery” than you (Christians) will have to rid the New Testament of “the great moral precepts and great sayings”, Jesus is said to have taught, such as “turn the other cheek” and “give to those who can’t repay you”. Likewise, you would also get rid of some questionable teachings, such as “hating your father, mother, brother, sister and yourself in order to follow Jesus” and “I have not come to bring peace to the world, but a sword”. Bottom line is, if you write off the Gospel of Thomas as heretical you are getting rid of almost all the sayings, good and bad, that your demi-god Jesus is supposed to have said, and having read the New Testament, you would not be left with much.

Examples

Gospel of Thomas Saying 55- “Jesus said: He who does not hate his father and his mother cannot be a disciple to me. And (he who does not) hate his brothers and sisters and take up his cross like me, will not be worthy of me.”

Luke 14:25-27- “Large crowds were traveling with Jesus, and turning to them he said:”If anyone comes to me and does not hate his father and mother, his wife and children, his brothers and sisters—yes, even his own life—he cannot be my disciple. And anyone who does not carry his cross and follow me cannot be my disciple.”

Gospel of Thomas Saying 44- “Jesus said: He who blasphemes against the Father will be forgiven, and he who blasphemes against the Son will be forgiven; but he who blasphemes against the Holy Spirit will not be forgiven, either on earth or in heaven.”

Mark 3:28-30 “Verily I say unto you, All sins shall be forgiven unto the sons of men, and blasphemies wherewith soever they shall blaspheme: But he that shall blaspheme against the Holy Ghost hath never forgiveness, but is in danger of eternal damnation. Because they said, He hath an unclean spirit.”

Matt 12:31-32- “Wherefore I say unto you, All manner of sin and blasphemy shall be forgiven unto men: but the blasphemy against the Holy Ghost shall not be forgiven unto men. And whosoever speaketh a word against the Son of man, it shall be forgiven him: but whosoever speaketh against the Holy Ghost, it shall not be forgiven him, neither in this world, neither in the world to come.”

Gospel of Thomas Saying 34- “Jesus said: If a blind man leads a blind man, they both fall into a pit.”

Luke 6:39- “And he spake a parable unto them, Can the blind lead the blind? shall they not both fall into the ditch?”

Gospel of Thomas Saying 11- “Jesus said: This heaven will pass away, and the one above it will pass away; and those who are dead are not alive, and those who are living will not die. In the days when you ate of what is dead, you made of it what is living. When you come to be light, what will you do? On the day when you were one, you became two. But when you have become two, what will you do?”

Mark 13:30-31- “Verily I say unto you, that this generation shall not pass, till all these things be done. Heaven and earth shall pass away: but my words shall not pass away.”

Gospel of Thomas Saying 2- “Jesus said: He who seeks, let him not cease seeking until he finds; and when he finds he will be troubled, and when he is troubled he will be amazed, and he will reign over the All.”

Luke 11:9-13-“And I say unto you, Ask, and it shall be given you; seek, and ye shall find; knock, and it shall be opened unto you. For every one that asketh receiveth; and he that seeketh findeth; and to him that knocketh it shall be opened. If a son shall ask bread of any of you that is a father, will he give him a stone? or if he ask a fish, will he for a fish give him a serpent? Or if he shall ask an egg, will he offer him a scorpion? If ye then, being evil, know how to give good gifts unto your children: how much more shall your heavenly Father give the Holy Spirit to them that ask him?”

and

Matt 7:7-11-“Ask, and it shall be given you; seek, and ye shall find; knock, and it shall be opened unto you: For every one that asketh receiveth; and he that seeketh findeth; and to him that knocketh it shall be opened. Or what man is there of you, whom if his son ask bread, will he give him a stone? Or if he ask a fish, will he give him a serpent? If ye then, being evil, know how to give good gifts unto your children, how much more shall your Father which is in heaven give good things to them that ask him?”

Gospel of Thomas Saying 10- “Jesus said: I have cast a fire upon the world, and see, I watch over it until it is ablaze.”

Luke 12:49-53- “I am come to send fire on the earth; and what will I, if it be already kindled? But I have a baptism to be baptized with; and how am I straitened till it be accomplished! Suppose ye that I am come to give peace on earth? I tell you, Nay; but rather division: For from henceforth there shall be five in one house divided, three against two, and two against three. The father shall be divided against the son, and the son against the father; the mother against the daughter, and the daughter against the mother; the mother in law against her daughter in law, and the daughter in law against her mother in law.”

and

Matt 10:34-39- “Think not that I am come to send peace on earth: I came not to send peace, but a sword. For I am come to set a man at variance against his father, and the daughter against her mother, and the daughter in law against her mother in law. And a mans foes shall be they of his own household. He that loveth father or mother more than me is not worthy of me: and he that loveth son or daughter more than me is not worthy of me. And he that taketh not his cross, and followeth after me, is not worthy of me. He that findeth his life shall lose it: and he that loseth his life for my sake shall find it.”

Gospel of Thomas 16- “Jesus said: Perhaps men think that I am come to cast peace upon the world; and they do not know that I am come to cast dissensions upon the earth, fire, sword, war. For there will be five who are in a house; three shall be against two and two against three, the father against the son and the son against the father, and they shall stand as solitaries.”

Matt 10:34-39- “Think not that I am come to send peace on earth: I came not to send peace, but a sword. For I am come to set a man at variance against his father, and the daughter against her mother, and the daughter in law against her mother in law. And a mans foes shall be they of his own household. He that loveth father or mother more than me is not worthy of me: and he that loveth son or daughter more than me is not worthy of me. And he that taketh not his cross, and followeth after me, is not worthy of me. He that findeth his life shall lose it: and he that loseth his life for my sake shall find it.”

Gospel of Thomas Saying 62- “Jesus said: I speak my mysteries to those [who are worthy of my] mysteries. What your right hand does, let not your left hand know what it does.”

Mark 4:10-12- “And when he was alone, they that were about him with the twelve asked of him the parable. And he said unto them, Unto you it is given to know the mystery of the kingdom of God: but unto them that are without, all these things are done in parables: That seeing they may see, and not perceive; and hearing they may hear, and not understand; lest at any time they should be converted, and their sins should be forgiven them.”

Gospel of Thomas Saying 68- “Jesus said: Blessed are you when you are hated and persecuted, and they will find no place where you have been persecuted.”

Luke 6:22-23- “Blessed are ye, when men shall hate you, and when they shall separate you from their company, and shall reproach you, and cast out your name as evil, for the Son of mans sake. Rejoice ye in that day, and leap for joy: for, behold, your reward is great in heaven: for in the like manner did their fathers unto the prophets.”

Gospel of Thomas Saying 77- “Jesus said: I am the light that is above them all. I am the all; the all came forth from me, and the all attained to me. Cleave a (piece of) wood; I am there. Raise up a stone, and you will find me there.”

John 8:12-20- “Then spake Jesus again unto them, saying, I am the light of the world: he that followeth me shall not walk in darkness, but shall have the light of life. The Pharisees therefore said unto him, Thou bearest record of thyself; thy record is not true. Jesus answered and said unto them, Though I bear record of myself, yet my record is true: for I know whence I came, and whither I go; but ye cannot tell whence I come, and whither I go. Ye judge after the flesh; I judge no man. And yet if I judge, my judgment is true: for I am not alone, but I and the Father that sent me. It is also written in your law, that the testimony of two men is true. I am one that bear witness of myself, and the Father that sent me beareth witness of me. Then said they unto him, Where is thy Father? Jesus answered, Ye neither know me, nor my Father: if ye had known me, ye should have known my Father also. These words spake Jesus in the treasury, as he taught in the temple: and no man laid hands on him; for his hour was not yet come.”

Gospel of Thomas Saying 38- “Jesus said: Many times have you desired to hear these words which I speak to you, and you have no other from whom to hear them. Days will come when you will seek me (and) you will not find me.”

Luke 17:22- “And he said unto the disciples, The days will come, when ye shall desire to see one of the days of the Son of man, and ye shall not see it.”

Sources

http://www.earlychristianwritings.com/thomas/

http://www.earlychristianwritings.com/

http://www.biblegateway.com/

*-The exact percentage is 84.2105% for those who thought I approximated by rounded up.

1 – For the full list of findings please email me at metroatheists@hotmail.com

Written by Joel

September 26, 2008 Posted by | Bible, Christianity, religion | , , , , , , , , , , , , , , , | 8 Comments

Beauty

“Everything which is in any way beautiful is beautiful in itself,
and terminates in itself, not having praise as part of itself. Neither
worse then nor better is a thing made by being praised. I affirm this also
of the things which are called beautiful by the vulgar, for example, material
things and works of art. That which is really beautiful has no need of
anything; not more than law, not more than truth, not more than benevolence
or modesty. Which of these things is beautiful because it is praised, or
spoiled by being blamed? Is such a thing as an emerald made worse than
it was, if it is not praised? Or gold, ivory, purple, a lyre, a little
knife, a flower, a shrub?”

- Marcus Aurelius, From book four of his Meditations.

Think about this quote when you think of the Biblical God.  Said to be beautiful, and yet demands our praise an worship.

- Chalmer

September 26, 2008 Posted by | philosophy, Qoutes | , , , , , , | Leave a comment

Artificial Intelligence

If the intelligence of the human mind is the product of the a physical mind, and thusly its actions dictated by physical laws, then it is reasonable to conclude that an artificial mind of equal physical capacity would be necessarily intelligent.  This is the principle of multiple realizability (1).  For any process that produces an affect, there is no reason to assume that said process is the only means of producing the very same affect.  That a form produces or infers a specific action does not logically imply that no other form could produce the same action.  While form must play a role in considering artificial intelligence, specific form does not need to be considered.
If the activities of the mind are not purely physical, and the emergent characteristics of the mind are not solely accounted for by said activities, then it would be unreasonable to conclude that an artificial mind would be necessarily intelligent.  If the latter case is true, it may be that, while some aspects of the mind are partly or purely immaterial, others, such as intelligence, may not be.  If the a specific causal relationship can be found between every aspect of intelligence and a physical process, then the hypothesis of the immaterial mind would be falsified.  The hypothesis is worthy of some consideration because it explains everything we have observed so far.  However, even prior to falsification the scientific community should not seriously consider the hypothesis.
If we presume existence of an immaterial mind we do so in spite of a large number of implausible, impractical, and confusing implications.  The presence of a forelife and afterlife are implied.  If the mind is, at least in part, separate from the physical realm then biological death should not eliminate it.  But once we begin to discuss the details of the nonphysical we find ourselves in the realm of pure speculation.  What parts of our mind are preserved and which are not, where did the mind come from, where will it end up, and where does it go?  These are only a few unanswerable questions.  Using the principle of Ockham’s razor we can remove rationalism and dualism from scientific inquiry because they raise more questions than they answer and most of the questions they raise can’t be answered at all (2).  So, if we disregard the idea of an immaterial mind we must acknowledge that an artificial mind is at least possible.
Humanity has created machines that can outwardly replicate many aspects of thought and reason.  For example, the chess playing Deep Blue computer observes its circumstances, the chessboard, and from a pool of all possible moves chooses the one most likely to ensure victory.  So, prompted by external circumstance, the machine acts to produce an effect.  However, this short description is as equally applicable to the rational process as it is of the falling of an anvil from the sky.  An anvil prompted by its external circumstance acts to produce an effect.  The action of the agent, i.e., of the machine, the anvil, or the person, is determined by the properties of that agent.  The machine acts to ensure victory because it is programmed to do so.  Deep Blue can no more deviate from this course of action by its own accord than an anvil can, by its own accord, deviate from descending from the sky.  In other words, the pool of potential actions for both of these non-human agents is limited by agents’ own physical form.
It is no longer appropriate to say that external circumstances determine an agents’ actions, but rather, that conditions both internal and external to the agent dictate the course of events.  However, the very same that has been said of the falling anvil and the Deep Blue computer can also be said of the human being.  So, lest we are content is saying an anvil has intelligence, further distinctions must be made in order to distinguish intellect from everything else.
I think it is important to make a distinction between indivisible and divisible properties.  Gravity, the tendency for objects of mass to accelerate towards one another, is an indivisible property of matter because the only possible alterations to it are elimination and opposition.  That objects might repel each other is the opposite of gravity, and therefore can not be termed equally.  That objects might have no affect on each other at all is a simple lack of gravity, and therefore it too can not be defines equally.  Any alteration of the anvil itself that would prevent it from acting consistently with the laws of gravity would change it so completely that it would no longer be possible to call it an anvil.  The ability to reason is also an indivisible property.  Similar to gravity, reason is process that mediates the relationship between causative conditions and resulting action or inaction.  However, the ability to reason towards survival, such as it might be in a human being, and the ability to reason towards victory, such as it is for Deep Blue, are divisible.  We can divide the application of reason with the intent of victory into two concepts.  The first concept is of invariable and indivisible reason, and the second concept is of the potentially variable intention of the act.  For the anvil, several hundred feet of this air between it and the grown can lead to only one action and only one intention, i.e., compliance with the law of gravity.  Any alteration to the intention of the act would force us to either alter the anvil so utterly that it no longer can be called an anvil, or to change or contradict the law of gravity.  The intention of the act in the context of reason, though, can be varied without altering the property of reason.  For Deep Blue, victory is the standard that the application of reason is meant to accomplish.  It is entirely possible that the intention of Deep Blue’s reason can be altered, say to the intention of loss, while simultaneously conserving its ability to apply reason and play a game of chess, however poorly.
Furthermore, the anvil’s actions determined completely by its unchanging internal characteristics.  However, Deep Blue was capable of learning by analyzing possible circumstances and altering its reactions to certain stimuli.  The programmers gave Deep Blue an intention and the potential to apply reason.  Upon analyzing numerous chess games, Deep Blue altered its strategy in order to achieve success.  With new information, Deep Blue was capable of self-directed changes to how it might react in the future.  No alteration of the properties of an anvil can alter the mechanism or the efficiency with which it succeeds in responding to gravity.  It must succeed, by virtue of its very nature, because success is the only option.  While Deep Blue might be enslaved to the goal of success, it is still realistically possible that it can fail to achieve that goal.  The anvil can’t fail or be made to fail, because gravity and matter are inseparable.
At this point, I still do not think it is reasonable to conclude that deep blue is intelligent.  Our intelligence might be an emergent byproduct of our mind’s processes, conferring no particular advantage, or it might the intended result of specific biological agents.  The latter case seems more likely because our consciousness seems to disregard the majority of our brain activity and seems restricted to certain regions of the brain.  This indicates that because deep blue has not been intentionally programmed with a conscious that it should not have one.  Until we understand the causal mechanism of consciousness, artificial intelligence will likely elude us.
Even if we can replicate all the intelligent processes of the mind, it still difficult to determine if our efforts have succeeded.  The Turing test, for example, identifies intelligence based on the symptoms of intelligence (1).  Multiple relizability, as discussed earlier, tells us that a single symptom could be causes by intelligent and non-intelligent processes.  Unless we understand the specific causal relationship responsible, we have no way of knowing if our machine is artificially intelligent.

- Chalmer

Citations
Lawhead, F. The Philosophical Journey: An Interactive Approach, Third Edition, McGraw Hill: New York, NY, 2006, pp 197-240

September 26, 2008 Posted by | philosophy | , , , , , , , , , , , , | 1 Comment

Summary analysis of Effects of Oxygen on Virulence Traits of Streptococcus Mutans

I wrote summary of this publication for my microbial genetics class. Probably pretty bland to most, but might as well post it.

Chalmer Wren

March 20, 2008

Summary analysis of Effects of Oxygen on Virulence Traits of Streptococcus Mutans

This is a summary of Effects of Oxygen on Virulence Traits of Streptococcus Mutans, by Sang-Joon Ahn, Zezhang T. Wen, and Robert A. Burne. The researchers sought to investigate the effects of aerobic and anaerobic conditions on the virulence traits of S. mutans. S. mutans, a gram-positive lactic acid bacterial species, is the primary contributor to tooth decay (1, p.266). This species of bacteria is localized in the crevices and pits of the teeth, which are a normal part of tooth topology. Tooth decay occurs as the result the acidic byproducts of the bacteria’s metabolic processes. Differential gene expression was first investigated by means of DNA microarray analysis, and the results validated by real-time quantitative RT-PCR on a subset of genes. Included among the genes which were down-regulated under aerobic conditions is gtfB, which encodes an enzyme critical to the process of biofilm production. The biofilm is an extracellular polysaccharide matrix which contributes to the virulence of S. mutans by protecting it from adverse environmental conditions and allowing it to adhere strongly to its substrate. Because the GtfB and GtfC proteins both play a significant role in biofilm production, the regulation of the genes encoding them were further investigated under aerobic and anaerobic conditions using a CAT assay and a western blot. Furthermore, the role of the VicK sensor kinase of a CovRS-like two component system (TCS) and the autolysin AtlA in S. mutans response to oxygen were also studied.

DNA microarray analysis is a powerful tool for studying genetics because it allows researchers to potentially monitor thousands of genes. Microarray analysis measures the relative amounts of RNA being produced via transcription by a cell (5, p.517). First, the desired RNA is isolated from the cells and purified. This RNA is then treated with reverse transcriptase and appropriate primers, resulting in the formation of a DNA RNA hybrid. The original RNA is then nicked by an enzyme so that DNA polymerase can utilize the RNA as a primer and then replace the RNA with DNA. The result of this process is called complementary DNA, or cDNA (5, p.769). The cDNA can then be cloned into a vector and labeled with a reporter gene. The cDNA is then allowed to hybridize with the immobilized DNA fragments of the microarray (5). This type of analysis indicates whether genes transcription is taking place, but is limited in its analysis because transcribed mRNA is not always necessarily translated. While useful for studying gene regulation at the transcriptional level, DNA microarray analysis reveals less about gene regulation at the level of translation.

DNA microarray analysis revealed that about 5% of the S. mutans genome displayed differences in genes expression, the majority of which were up-regulated under aerobic conditions. Table 3 lists the differentially expressed genes and the magnitude of difference in expression of each gene between aerobic and anaerobic conditions. Also listed in Table 3 are the P-values, which is an indication of the reasonableness of acquired measurements. Under aerobic conditions 83 genes displayed up-regulation while only 23 genes displayed down-regulation. Figure 1 shows the distribution of genes whose expression was altered by the availability of oxygen. Up-regulated genes were placed in five categories. 28 of the up-regulated genes were classified as hypothetical, unassigned, or unknown, 23 were classified as encoding energy metabolism proteins, 9 encoding signal transduction proteins, 8 encoding transport and binding proteins, and 4 encoding cellular process-proteins.

Of the 16 genes displaying a 10 to 42 fold difference in expression, many are either established or predicted to encode mutacins, which are the bacteriocins of S. mutans. Mutacins are exported by the cell with the intention of limiting competition from closely related species by killing them or interfering with their growth. The researchers postulate that the increased production of mutacins is an effort to limit competitive pressures prior to biofilm maturation. The fact that biofilm formation is a significant contributor to the virulence of S. mutans, and that biofilm formation is inhibited by aeration, is consistent with the observed increase in mutacin production. Lack of a biofilm exposes the S. mutans to a significant number of environmental pressures, including competition from other organisms in the environment, thus dedicating cell resources to creating mutacins would be advantageous.

bip, which encodes a reputed bacteriocin immunity protein, was up-regulated 18-fold. The Bip protein has been implicated in providing resistance not only to bacteriocins, but to a variety of other environmental pressures. These include, but are not limited to, antibiotics, metals, and attacks from the host organisms’ immune system. As mentioned earlier, exposure to inhibitory components of the environment increases when the formation of the protective biofilm is compromised. Therefore the necessity to produce Bip and related proteins becomes increasingly important.

comD, a gene which codes for the histidine kinase of a TCS and functions to increase the expression of com genes via the process of induction, was up-regulated 2.3 fold under aerobic conditions. The comD gene also functions in the development of competence, which is the ability of a cell to import naked DNA from the extracellular space (1, p. 278). The product of the comC gene is a competence stimulating peptide (CSP), the receptor of which is the integral membrane protein called ComD with histidine kinase activity. When ComD is bound to extracellular CSP, the histidine kinase activity of ComD is activated. This leads to altered expression of gene associated with competence (8). This particular instance of regulating gene expression is part of the comC quorum-sensing system (ComCDE), which describes an intracellular communication system of bacteria which alters gene expression in response to population density (9). Previous studies have also shown ComCDE to play a role in appropriate biofilm formation for S. mutans (9).

Previous studies have established that aerobic conditions prompt lactic acid bacteria to resort to a heterofermentative metabolism. The same should also be true for S. mutans. As expected, differential gene expression associated with metabolism was observed in the genes associated with the partial tricarboxylic acid cycle, which will hereafter be referred to as the Szent-Györgyi-Krebs cycle to honor its principle contributors, of S. mutans. The genes associated with the Szent-Györgyi-Krebs cycle were up-regulated. As well, up-regulation of enzymes such as NADH oxidases and pyruvate formate lyase were also observed. NADH oxidase enzymes allow the organism to metabolize oxygen, a function that is important for survival in aerobic environments. Pyruvate dehydrogenase oxidizes pyruvate, which is a product of glycolysis, to form acetyl-CoA, an important precursor of the Szent-Györgyi-Krebs cycle (4). .

Increased production of CcpA, a DNA binding protein, was also indicated by microarray analysis. One aspect of metabolism affected by cultivation in the presence of oxygen is glycolytic rates, the variation of which would lead to a change in the concentration glycolytic intermediates. CcpA is part of a global regulatory system which, in response to the variation of in the concentration of glycolytic intermediates, alters the transcription of genes encoding enzymes of the glycolytic pathway, the Szent-Györgyi-Krebs cycle, and enzymes involved in carbohydrate acquisition and catabolism. Additionally, CcpA has been shown to play an important role in regulating expression of genes whose products are used in biofilm formation. These genes are gtfB, gtfC and ftf, which encode enzymes needed for the production of a major component of the biofilm, called exopolysaccharides.

As mentioned previously, genes affected by the regulatory role of CcpA include those corresponding to the importation carbohydrates from the extracellular environment. So, the observation of up-regulation in genes associated with the phosphophenopyruvate:sugar transerase system (PTF) and the ATP-binding cassette is consistent with what is known about the function of CcpA. The affect aeration has on the production of CcpA and its regulatory role in the expression genes associated with the acquisition of carbohydrates and their subsequent metabolism is an important observation because it indicates a response by S. mutans at the level of transcription.

Of the down regulated genes, 8 are classified as hypothetical or unknown, 4 are involved in transport and binding, 2 in energy metabolism, 2 cellular processes, and 2 in protein fate. One important observation was the up-regulation of relP in conjunction with the down-regulation of genes involved in amino acid metabolism. The product relP catalyzes the synthesis of (p)ppGpp, whose suggested function is to alter metabolism. (p)ppGpp compromises expression of genes needed for cell growth in order to produce an increased expression of genes which aid in stress tolerance and amino acid synthesis.

Cultivation in aerobic conditions yielded 3.3 fold down-regulation of gtfB. Gtfb is an essential enzyme needed for the production of the glucan polymers, a component of the biofilm. These glucan polymers are water insoluble and adhesive, properties which contribute significantly to the functions of biofilms. According to the researchers, the down regulation of gtfB may be a factor in reducing S. mutans’ ability to form biofilms. gtbC, a gene which is cotranscribed with gtfB, was neither up-regulated or down regulated according to DNA microanalysis. Because of their significance in establishing a biofilm, the expression of these genes was further investigated.

Figure 2 shows a CAT assay whose results establish the same trend as the microarray analysis, which is the up-regulation gtfB and a lack of differential expression for gtfC. The CAT assay was produced by fusing the gtfB and gtfC genes with a reporter gene in a process described in more detail in the subsequent paragraph, and then inserted as a single copy into strain UA159, which is wild type, to produce two new strains. The resulting strains, indicated in table 1, were TW54 and TW55. TW54 contained gtfB promoter fusion, while TW55 contained the gtfC promoter fusion. The reporter gene for this analysis is the chloramphenicol acetyltransferase (cat) gene. Chloramphenicol is an antibiotic that functions by binding the ribosome and inhibiting the process of translation. The cat gene gives a bacterium a resistance chloramphenicol (1). Following cultivation of cells, the relative amount of protein produced by cat can be measured. The CAT assay essentially measures effectiveness of a genes promoter, within a given environmental context. The original gene is replaced with the reporter, and the reporters production measured.

In order to construct the reporter gene fusion, PCR was performed on a fragment of DNA containing the promoter regions and ribosome binding sites (RBS) for each gene. The primers used for this process are listed in Table 2. In order to produce the expression of the reporter gene, the products of PCR were cloned into plasmid pU1. Cloning a gene into a plasmid first requires that the plasmid is digested to a linear DNA molecule by a restriction endonuclease. The restriction endonucleases used in this part of the experiment were BamHI and PstI, which create a staggered or sticky end (5). They are capable of creating a staggered break because they recognize sequences with twofold rotational symmetry. The resulting staggered unpaired ends read the same in the 5’ to 3’ direction and this can pair with and be ligated to any other piece of DNA digested by the same restriction endonuclease (5,). The transcriptional fusions were then released by digestion with SmaI and HindIII, also restriction endonucleases, and inserted into pBluescript KS(+) which had been digested by the restriction. endonucleases Eco-RV and HindIII. The resulting plasmid was then digested by restriction endonucleases SmaI and HincIII. HincIII and SmaI differ from the restriction nucleases previously described because they create blunt ends instead of sticky ends. Blunt ends lack the unpaired bases of staggered ends and this have a much lower affinity for the ends of other DNA strands (6,7). The DNA fragments were then purified and ligated into the integration vector, which was then inserted into the gtfA locus of strain UA159 to create strains TW54 and TW55. The results of the CAT assay displays the same trend observed in the DNA microarray analysis, which was that gtfB was up-regulated and gtfC was unaffected.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and a Western blot was performed of protein fractions from S. mutans in order to compare the localization of GtfB and GtfC proteins of cells cultured in aerobic to those cultured in anaerobic conditions. Sodium dodecyl sulfate (SDS) is a detergent which binds to protein in an amount approximately proportional to the molecular weight of that protein and gives it a net negative charge. Electrophoresis in the presences of SDS separates proteins on the basis of mass(4, p.92). The results of the SDS-PAGE and Western blot, to be discussed later, are displayed in Figure 3. GtfB and GtfC were detected using a polyclonal rabbit antibody. The protein fractions used for SDS-PAGE and Western blot were obtained in a series of steps, the first of which was centrifugation and two washes with Tris-buffered saline. Supernatant proteins were obtained by passing the supernatant fluid through a filter and precipitating the proteins with trichloroacetic acid. Whole cell lysates were obtained by homogenization in an SDS boiling buffer with glass beads in a Bead Beater. Surface associated proteins were obtained by incubating cells in a 4% SDS solution for 30 minutes. The soluble fraction, containing soluble proteins of the cytosol, was obtained by centrifugation of the homogenized cells, which separates soluble and insoluble proteins. Soluble proteins remain in the supernatant, and the pellet is resuspended in SDS boiling buffer to create the insoluble fraction.

Each fraction, the sum of which comprise the whole cell lysate, indicates the amount of GtfB or GtfC within different parts of the cell, this providing data about protein localization. For each fraction, the 4% SDS extract isolates the proteins associated with the cell surface, the soluble fraction contains proteins of the cytosol, and the supernatant fraction contain proteins exported by the cell. The results in Figure 3 indicate that for the 4% SDS extracts (B), the soluble fraction (C), and the insoluble fraction (D), the amount of GtfC as indicated by the intensity of the band was greater in magnitude for the aerobically cultivated cells than for the cells grown in anaerobic conditions. In the supernatant fraction (C), the relative amounts of GtfC were equivalent in magnitude. While Western blot results of GtfB are consistent with the DNA microarray analysis and the CAT assay, it indicates an overall increase in the amount of produced GtfC, which neither of the previous two methods indicated.

Unlike the CAT assay, SDS-PAGE and the subsequent Western blot used a polyclonal antibody to detect GtfC and GtfB directly. The variation in methodology might play a role is the variation observed results.

This study also showed that VicK sensor kinase of the CovRS-like TCS plays a role in the response of S. mutans to aerobic environments. The sensor kinase of a TCS detects changes in the environment and when the appropriate conditions are satisfied it, as its name would imply, phosphorylates protein of the cytosol which plays a role in regulating gene expression (10). The CovRS TCS of S. mutans is a regulatory system which regulates the expression of glucosyltransferase genes, ftf, and a gene encoding a glucan-binding protein (11). Analysis, discussed in further detail in the subsequent paragraph, revealed inactivation of the genes for VicK restored the ability of S. mutans to form biofilms in the presence of oxygen.

In a recent study performed by the investigators it was observed that the VicK-deficient strain, indicated as vicK-NP in Table 1, produced elevated levels of GtfC. VicK is a component of the VicRKX TCS. A PAS domain, which was recently reported to be present in the VicRKX TCS, is a potential sensor for redox potential. This is important because redox potential of mature biofilms differs from that of early biofilms. When subjected to SDS-PAGE analysis, both GtfB and GtfC on the vicK-NP strain an compared to UA159, the amount of both proteins had increased in the soluble and insoluble fractions from vicK-NP strain, while, in the same strain, a reduction in the levels of both proteins was observed for the supernatant fractions. The investigators also measured the levels of mRNA for vicK-NP, the results of which are consistent with the SDS-PAGE analysis in that they display and increase in transcription of gtfC. Figure 4A displays the results of the SDS-PAGE comparison of UA159 and vicK-NP, and Figure 4B displays the results of real-time PCR, used to measure amount of transcribed mRNA.

In addition to the VicK sensor kinase, this study also investigated the role of the AtlA autolysin pathway and its role in meadiating S. mutans’ response to oxygen. Like the VicK sensor kinase, inactivation of the genes for AtlA restored the bacteria’s ability to form biofilms in aerobic conditions. The AtlA autolysins pathway plays an essential role in the formation and maturation of the biofilm, and the regulation of its associated genes by the ComCDE regulatory system has been implicated in other studies (11).

This study established that exposure to oxygen has a profound effect on the virulence of S. mutans, especially in regards to biofilm formation. As well, it was demonstrated that VicK sensor kinase and the AtlA autolysin pathway both play an important regulatory role with gene expression associated with biofilm was established.

Bibliography

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3. National and Maternal Child Resource Health Center. What is Streptococcus Mutans? 2006. http://www.mchoralhealth.org/OpenWide/mod1_2.htm (accessed on March 20, 2008.)

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9. Li, Y.; Tang, N.; Aspiran, M.; Lau, P.; Lee, J.; Elle, R.; Cvitkovitch, D. A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm. Journal of Bacteriology. [Online] 2002. 184. 2699-2708. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135014 (accessed March 19, 2008)

10. Forsyth, M.; Cao, P.; Garcia, P.; Hall, J.; Cover, T. Genome-Wide Transcriptional Profiling in a Histidine Kinase Mutant of Helicobacter pylori Identifies Members of a Regulon. Journal of Bacteriology. [Online] 2002. 184. 4630-4635. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135264 (accessed March 19, 2008)

11. Burne, R.; Ahn, A. Effects of Oxygen on Biofilm Formation and the AtlA Autolysin of Streptococcus mutans. Journal of Bacteriology. [Online] 2007, 89, 6293-6302. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1951938 (accessed March 19, 2008)

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