The purpose of the experiment described by Griffin et al. (1975) is to identify the transport mechanism used by tetanospasm (tetanus toxin) to reach the CNS. This information is important because the first step to fully understanding how a substance interacts with the body is to find its means of transportation. In addition, discovering tetanospasmin's transportation patterns may give incite to other toxins and may allow researchers to control tetanospasmin's activity and even the administration of tetanospasmin in future treatments if the toxin can be modified. Previous researchers have claimed that tetanospasmin transports either through the peripheral nerve endings, intraaxonal transport or simply through the vascular system and lymphatic system (Griffin et al. 1975). In order to perform autoradiography on tetanospasm, a radioisotope (I125) was injected to visually aid the researchers with identifying where tetanospasm was found subsequent to the injection of the toxin (Griffin et al. 1975). An axonal crush was induced in the nerve (an end reference point), specifically on the axon, to view the proximal-distal migration of tetanospasm (Griffin et al. 1975). Upon injecting the mice with tetanospasm, the labeled toxin was found to accumulate at the distal side of the crush and displayed a dense accumulation of silver grains near the axonal crush(Griffin et al. 1975). Despite this fact, tetanospasmin was not found in any of the extraaxonal components in the nerve, such as the Schwann cells or the blood vessels. The authors also observed silver grains in the distal end of the crush, but no traces of silver grains were found in the proximan region to the crush. This observation led to the conclusion that the transportation occurred in a retrograde fashion, which is the opposite direction of an action potential in order to reach the cell body (Griffin et al. 1975). The author concluded from the previous theories suggested by scientist, that tetanospasmin's mechanism of travel is through an intraaxonal fashion, since there was accumulation of the toxin only within the axon. Furthermore, the authors also suggested that tetanospasmin travelled only in a single direction, since silver grains were only viewed on one side of the crush, more specifically on the distal side, which confirms that tetanospasmin travels in a retrograde fashion to reach its final destination (Griffin et al. 1975). Lastly, tetanospasmin could not enter from any area of the nerve, rather it must enter through the neuromuscular junction in order to cause the effects associated with it (Griffin et al. 1975).
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In 1975 Griffin and associates unraveled the mystery of tetanospasmin's direction and intraaxonal transportation mechanisms. Their discovery was very important, however; they only focused on where the toxin entered the nerve axons and what mechanism it used in order to reach its destination once it entered the nerve, while keeping a blind eye to tetanospasmin binding domain, which allows the toxin to enter the nerve. In 1992 Jane et al took the next step into fully understanding tetanospasm through the analysis of its specific binding domain.
The purpose of the experiment described by Jane et al (1992) is to identify and locate the exact binding domain of Hc (heavy chain). The importance of this experiment is to develop further knowledge of tetanospasmin and gain the knowledge of the exact location of the toxin's binding region. Identifying the exact binding domain allows future researchers to look at the exact mechanism Hc uses to enter into the cell and may allow researchers to intercept tetanospasmin if needed. In order to study the Hc of tetanospasmin, the authors required an ample amount of the Hc to experiment on. The first step in attaining an ample sample size is to purify, isolate and label (using I125) Hc from a bacterial culture (Jane et al, 1992). Once the authors isolated the Hc sample they ran the sample through PCR (Polymerase Chain Reaction) on a template plasmid (SS1261), which contains the sequence of the tetanospasmin (Jane et al, 1992). Upon amplifying the gene through PCR the gene was transcribed into RNA to allow the authors to quickly change the mRNA strand into its complementary protein. Therefore the authors injected tetanospasmin in vivo to achieve multiple mRNA copies of the heavy chain (Jane et al, 1992). Once enough copies were retrieved they cleaved the Hc in different regions (including the amino side of the protein and the carboxyl side) to find the amount of nucleotides that are crucial for Hc to function properly and how many nucleotides were needed to attach to its receptor (Jane et al, 1992). The cleaved Hc copies were run though SDS PAGE and autoradiography to view the amount of amino acids that were cleaved (Jane et al, 1992). The authors observed that cleaved Hc proteins on the carboxyl side functioned properly when only 5 amino acids were cleaved but upon cleaving 10 or more amino acids from the carboxyl side, the function of the whole protein was abolished (Jane et al, 1992). Furthermore, upon cleaving the amino acids from the amine side, the Hc protein maintained its function even when 263 amino acids were cleaved (Jane et al, 1992). This information allowed the authors to observe the importance of the carboxyl side of the Hc, but it did not provide direct evidence that carboxyl region is the region that is bound to the gangliosides (receptor for tetanus toxin). Therefore, the authors synthesized an antibody that stimulates the binding region of the gangliosides to view if there was an interaction between the C terminus and the gangliosides (Jane et al, 1992). The authors observed that there was no interaction even with very high concentration of the C terminus. Moreover, the importance of the carboxyl end was already viewed so the only suggestion left is that C terminus may cause the toxin to have the proper structure to bind (Jane et al, 1992). The authors placed all the Hc proteins into a solution containing trypsin in order to view if the copies would be cleaved. The authors observed that all the truncated Hc copies were degraded while the non-truncated protein retained its shape (Jane et al, 1992). The researchers concluded that the C terminus of the Hc region of the tetanospasm was extremely important for keeping the shape of the toxin, and if even 10 amino acids were cleaved from the carboxyl side, the toxin would not be able to enter any cells.
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According to Jane et al, 1992, the Hc (heavy chain) at the carboxyl side in tetanospasmin is important for tetanospasmin to invade the gangliosides. However, the method of entry by the TeNT (tetanus toxin) was still unknown (Matteoli et al, 1996). Matteoli and his associates went forth to discover the mechanism TeNT uses to enter the cell.
The purpose of the experiment performed by (Matteoli et al, 1996) is to discover the mechanism which is used by TeNT to penetrate its target cells. This discovery is important because it may allow subsequent researchers to identify a possible mechanism used by toxins to invade the cell. In order to view the toxin in the cell, the authors added a fluorescent dye, Texas red, to TeNT (Matteoli et al, 1996). The TeNT that annealed to the dye (TeNT-TR) was viewed and retrieved after all the samples were run through the chromatography column (Matteoli et al, 1996). In addition, the authors were required to use immunocytochemistry to label their target cells (Matteoli et al, 1996). The target cells inside the neuronal cells (SV2 and VAMP-2) that are cleaved by TeNT to cause paralysis were also labeled using a dye and antibodies with fluorescence attached to them (Matteoli et al, 1996). This procedure was used by the authors to allow them to view the amount of the target cells that are present in the cell. In addition, the initial concentration of the two substrates were counted and recorded (Matteoli et al, 1996). The authors used 6 different neurons (labeled A-F) and added the same substance to the neurons with adjacent letters (A and B with the same addition, C and D with the same addition, etc). The neurons A-D were immersed in a solution that contained KCl and neurons E and F were placed into a control media (without any addition of extra substrates such as KCl). Furthermore, calcium was added to neuron A and B, in order to view, which substance (KCl or calcium) is the limiting reagent that is needed by TeNT to penetrate the neurons (Matteoli et al, 1996). Lastly a counterstain was used on neurons A, C and E, since the counterstain would always show fluorescence even if TeNT did not penetrate the cell, because the counterstain binds to SV2 markers which should be present in the cultured cells. The authors observed that only neuron B displayed a distinct color from the non-control groups, while all the control groups (neurons A, C and E) displayed fluorescence. The authors concluded that tetanus toxin is taken up at nerve endings, since potassium is a substance that promotes exocytosis through vesicles and uptake of vesicles through endocytosis (Matteoli et al, 1996). In addition, the authors found that calcium and KCl are both essential for the uptake of the toxin and the mechanism, and both are needed to be found close to the terminal of a neuron in order for tetanus toxin to enter the cell and cause its effects (Matteoli et al, 1996). Furthermore, one could extrapolate that tetanus toxin enters the cell through active transport, due to the calcium and KCl that is needed to enter the cell (both which are active ingredients to active endocytosis).
Tetanospasmin is known for its toxic effects and its ability to cause an individual to engage in whole body massive muscle spasms, which causes an individual to die in a painful manner. Throughout all the findings, the researchers have shown that tetanospasmin binds to its receptor on gangliosides through its heavy chain (Jane et al, 1992) and enter the cell through synaptic vesicles using calcium and KCl (Matteoli et al, 1996), while the light chain has been discovered to cause the toxic effects it is associated with (Li et al, 1998). Since the toxicity of tetanus is associated with the Light chain, the heavy chain's function is still not fully known, in 2004 ChaÄ±¨b-Oukadour and his associates set forth to view the effects of only the heavy chain of the tetanus toxin.
The purpose of the experiment performed by (ChaÄ±¨b-Oukadour et al, 2004) was to identify the effects of the C-terminus on the heavy chain of tetanus toxin (Hc-TeTx) and to compare the effects of Hc-TeTx to an already known effect of tetrazolium salts. The importance of this experiment lies in discovering the function of the Hc-TeTx. If the function of Hc-TeTx parallels the tetrazolium salts (to combat neurodegeneration), the authors could introduce an alternative way in which tetanus toxin is viewed by other researchers and the authors may be able to introduce new alternative treatments for individuals with neurodegenerative conditions. In order to test Hc-TeTx Cerebellar granule neurons (CGN) were isolate from mice and isolated through immunoprecipitation and centrifugation of the CGN (ChaÄ±¨b-Oukadour et al, 2004). Subsequent to isolating the CGN the cells were lysed and the supernatant was blotted on a Western Blot and the proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The antibody which were used during the western blot corresponded to antibodies that detect the presence of p21Ras-GTP, which begins the intracellular signals for activating many substrates such as ERK and PKB, and antibodies specific for ERK or Akt/PKB (both of which are anti-apoptotic signals which inhibits apoptosis in the cell) (ChaÄ±¨b-Oukadour et al, 2004). As a final confirmatory check the authors ran a MTT reduction assay which allows the researchers to view the CGN cells under a phase-contrast microscope, which allows them to determine if a cell is going through apoptosis (through an increase of fragmented DNA) or if a cell remains intact. During this test they placed the neurons in a medium containing 25 mM KCl (which allows neurons to survive) and placed Hc-TeTx in the same location. Furthermore, the neurons were placed in a 5 mM KCl medium, 25 mM KCl and a medium which contained 5 mM KCl and Hc-TeTx. In the medium which contained 25 mM KCl the cell displayed little to no fragmented DNA, while in the 5 mM KCl medium more than 15% of the cells displayed fragmented DNA. Additionally, with the addition of both Hc-TeTx and 5 mM KCl the cells displayed 10% fragmentation (ChaÄ±¨b-Oukadour et al, 2004). Moreover, the author also observed that there was a large increase in the activity of p21Ras-GTP, Akt/PKB and ERK when Hc-TeTx was introduced into the solution. The authors concluded that Hc-TeTx has a direct effect on the apoptosis of the neurons in the medium which causes their degeneration. Hc-TeTx induced an increase in the activity of p21Ras-GTP, which the authors believe led to the increase of Akt/PKB which directly affects the cell cycle and allows the cells to live longer, even under conditions where they are malnutritioned (ChaÄ±¨b-Oukadour et al, 2004).
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ChaÄ±¨b-Oukadour and associates discovered that Hc-TeTx increases the amount of apoptotic enzymes/kinases which allow the neuron to live longer. The apoptotic effects viewed by ChaÄ±¨b-Oukadour et al were centered on CGN cells, which does not have a lot of real life application. Their discovery was very important for building the ground work for a very important real life application. Mendieta et al used ChaÄ±¨b-Oukadour et al's approach and shifted their sight towards the application of Hc-TeTx in MPP+ rats, which exhibit dopaminergic neuron degeneration. The lack of dopiaminergic neurons causes the condition of Parkinson's disease (PD)
The purpose of the experiment described by Mendieta et al. (2009) is to analyze the effects of administering the Hc-TeTx (C terminus of the heavy chain of tetanus toxin) to MPP+-treated rats and to observe its effects on the dopamine levels. This study is important because MPP+-treated rats exhibit the symptoms as patients with Parkingson's disease. This is because MPP+ treated rats have very low levels of dopamine due to the death of their dopaminergic neurons Mendieta et al. (2009). The first step to finding a cure to Parkingson's disease is to find a substance that will halt the death of dopaminergic neurons . The authors selected rats at random and injected the rats with either Hc-TeTx or MPP+ or Hc-TeTx and MPP+ in the striatum. The purpose of the three groups is to have 2 control groups - the MPP+-treated rats and Hc-TeTx-treated rats. Both these groups would allow the authors to maintain a clear image of the effects that both of these substances cause to a perfectly healthy rat. In addition, the injection of both Hc-TeTx and MPP+ was used to illustrate the effect of Hc-TeTx on mice that were inflicted with MPP+. They observed that the rats that were merely injected with MPP+ displayed an exponential decrease in motor functions, as expected, while the rats injected with Hc-TeTx demonstrated normal motor functions. Hence, the authors were able to go forth with the experiment and combine both Hc-TeTx with MPP+ in the rats, knowing that Hc-TeTx does not have any harmful effects on rats, while MPP+ displayed to have deleterious effects on the rats. Upon injecting both substances, the rats were run through the ipsilateral turns test (measures turns/min) and the researchers noted that the rats with Hc-TeTx and MPP+ turned half as many times as the MPP+ rats. This clearly displays that the rats given the Tetanus toxin heavy chain had much better motor control than the MPP+-treated rats. Furthermore, many other tests were used and all of the tests displayed similar results to the ipsilateral test. Additionally, dopamine levels were also measured in order to view if this Hc-TeTx would only treat the symptoms; as the current drugs prescribed for Parkingson's disease patients do, or if Hc-TeTx affected the direct cause of the death of the dopaminergic nerves. The researchers observed that the dopamine levels for Hc-TeTx MPP+-treated rats was almost as high as the control group while the MPP+-treated rats had only 60% of the dopamine located in the striatum. Overall the authors concluded that the C terminus of the heavy chain from the tetanus toxin may be used in order to slow the degeneration of patients with Parkingson's disease and can be very effective in the case of early diagnosis. The next step towards using Hc-TeTx as a drug is to test Hc-TeTx on different animals to confirm that the effects are similar. This is because some animals react differently to substances that humans do.
Throughout many different experiments the researchers concluded that tetanospasmin enters the cell using its C-terminus on the heavy chain through synaptic vesicles and travels up the axon in a retrograde fashion (Matteoli et al, 1996). Furthermore, the heavy chain specifically contained the domains that allowed tetanospasmin to enter the cell and cause its effects (Jane et al, 1992). Fortunately, the toxicity that is caused by tetanus resides on its light chain which was excluded from the tetanospasmin that was administrated to the rats/mice in most of the experiments (Li et al, 1998). The authors concluded that tetanospasmin not only inhibits neurons from degenerating through activating many apoptotic cascades through activation of kinases and enzymes, but it also can target specifically the dopaminergic neurons, which degenerate in PD patients (Mendieta et al. 2009). Currently, Parkingson's disease patients lack the ability to make a full recovery, but with all the new research put forth and the discoveries being made, it will only be a matter of time before Parkinson's disease will be a condition in the past.
(http://www.ncbi.nlm.nih.gov/sites/entrez, called Expression and Characterisation of the Heavy Chain of Tetanus Toxin:
Reconstitution of the Fully-Recombinant Dichain Protein in Active