Browsing by Subject "Organismal Biological Physiology"
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Item Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur EisenbergThe AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.Item Positive Regulation of Acetate Metabolism and Motility by the RNA-Binding Protein CsrA in Escherichia coli(2000-08-01) Wei, Bangdong L.; Jerry Simecka; Ming-Chi Wu; Stephen R. GrantWei, Bangdong L., Positive Regulation of Acetate Metabolism and Motility by the RNA-binding Protein CsrA in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), August, 2000, 118 pp., 5 tables, 19 illustrations, bibliography, 175 titles. The carbon storage regulatory (Csr) system consists of a small RNA-binding effector protein, CsrA, and non-coding RNA, CsrB. CsrA acts as a global regulator and modulates specific mRNA stability in Escherichia coli. It regulates central carbon metabolism, physiology, and cell surface properties on a broad scale. In this study, the regulatory roles of csrA in acetate metabolism and motility were examined. The csrA gene was demonstrated to positively regulate acetyl-CoA synthetase and isocitrate lyase, while it did not affect phosphotransacetylase, isocitrate dehydrogenase, or citrate synthase. As a result, growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media. Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. The TCA cycle intermediates or pyruvate, but not glucose, galactose or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. Apparently, central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism are combined to cause metabolic stress by depleting the TCA cycle. The csrA gene was essential for motility and flagellum biosynthesis. Further studies elucidated the molecular mechanism by which CsrA positively regulates flagellum synthesis. Purified recombinant CsrA protein, which was isolated as a ribonucleoprotein complex consisting of one single CsrB molecule and ~18 CsrA subunits, directly stimulated the coupled transcription-translation of flhDC::lacZ in S-30 extracts and bound specifically to the 5’ non-coding segment of flhDC mRNA in mobility shift assay. The steady state level of flhDC mRNA was higher and its half-life was ~3-fold greater in a csrA wild type versus a csrA::kanR mutant strain, as shown by RT-PCR. Thus, CsrA is able to stimulate flhDC gene expression by a post-transcriptional mechanism that resembles its function in repression.Item Resistance of Bacillus Subtilis Spores Lacking Either Nucleotide Excision Repair or Spore Photoproduct Lyase to Ultraviolet (UV) Radiation from Artificial or Natural Sources(1996-06-01) Xue, Yaming; Tony Romeo; Ming-Chi Wu; Wayne L. NicholsonXue, Yaming, Resistance of Bacillus subtilis spores lacking either nucleotide excision repair or spore photoproduct lyase to ultraviolet (UV) radiation from artificial or natural sources. Master of Science (Biomedical Sciences), June 1996, pp., 4 tables, 12 illustrations, references, 38 titles. Exposure of bacterial spores to UV radiation causes the accumulation of a unique pyrimidine dimer in the DNA, “spore photoproduct” (SP). In Bacillus subtilis, two distinct DNA repair pathways are used for removal of SP: general nucleotide excision repair (the uvr parthway), or the SP-specific enzyme SP lyase (the spl pathway). Spores of four strains of Bacillus subtilis differing in their repair capabilities were irradiated under either artificial or solar UV. To determine the biologically-relevant cumulative UV dose under each irradiation condition, a sporocidal dosimeter was constructed. The results showed: (i) Both uvr and spl pathways contributed to the survival of spores under all tested conditions. The spl pathway was more efficient than uvr pathway in repairing the DNA damage caused by UV-C and solar UV-A, but no significant difference was noted in repairing DNA damage caused by UV-B or full-spectrum solar UV. (ii) Exposure of spores to solar UV can cause cellular lethal damage which is reparable by neither repair pathway.Item The Influence of Aging and Glycation on Protein-Thiol Mixed Disulfides in the Eye Lens(1994-06-01) Dickerson Jr., Jaime E.; McConathy, Walter J.; Yorio, Thomas; Lou, MarjorieJaime E. Dickerson, Jr., The Influence of Aging and Glycation on Protein-Thiol Mixed Disulfides in the Eye Lens. Doctor of Philosophy (Biomedical Sciences), June, 1994, 163 pp., 9 tables, 28 illustrations, bibliography, 116 titles. The human lens is continually growing. As new cells are formed they differentiate into fiber cells which have no organelles, no protein synthesis or turnover. Lens protein aging involves formation of very large aggregations and insoluble complexes. These are held together through disulfide linkages. Reduced gluthathione (GSH) is present in high concentrations. The oxidized form, (GSSG), (5% of the total) can form mixed disulfides with proteins. This can destabilize the protein conformation. Accumulation of mixed disulfides may increase the potential for further modification. The participation of a PSSG (protein/gluthathione mixed disulfide) in the formation of a protein-protein disulfide becomes increasingly likely. The purpose of this work is to document PSSG and protein-cysteine mixed disulfide (PSSC) accumulation in human lenses (through eight decades), and to identify a third mixed disulfide discovered in this research. The free thiol molecules GSH and cysteine were also quantitated for normal and cataractous lenses. Glycation may alter conformation similar to mixed disulfides and potentiate mixed or protein-protein disulfide formation. This model was evaluated two ways. First, purified alpha crystalline was incubated with ascorbate and conformational changes were evaluated with CD spectroscopy. Second, rat lenses were cultured under high sugar conditions to determine if the resulting glycation influenced the level of mixed disulfides. Conversely, the effect of prior mixed disulfide formation on the extent of glycation in another purified crystalline, gamma, was evaluated. The results indicate: GSH declines in the lens with age, cysteine exists in the lens albeit at relatively low levels, PSSG shows a triphasic pattern of accumulation, PSSC accumulated linearly with increasing age, the existence of a third mixed disulfide species, gamma glutamylcysteine mixed disulfide, detected in old or cataractous lenses, has been confirmed, glycation by ascorbic acid alters α- crystalline secondary structure, the influence of glycation is minimal on mixed disulfide formation, mixed disulfide formation affects glycation of gamma crystalline.Item The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-Dependent Protein Kinase II in Pancreatic β-Cells(1998-06-01) Krueger, Kimberly A.; Richard Easom; Rafael Alvarez; S. Dan DimitrijevichKrueger, Kimberly A., The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-dependent Protein Kinase II in Pancreatic β-cells. Doctor of Philosophy (Biomedical Sciences), June, 1998, 165 pp., 35 illustrations, references, 259 titles. Increasing evidence supports a physiological role for calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from pancreatic β-cell. While it has been previously demonstrated that CaM kinase II is activated by glucose in isolated rat islets implicating this enzyme in the secretion process, its cellular targets are unidentified. Potential candidates would likely exhibit strong binding to the enzyme, an association with the cytoskeleton, or an involvement in the secretion process. Based on these criteria, the following study represents an evaluation, in situ, of two proteins to function as substrates for CaM kinase II. Microtubule-associated protein, MAP-2 is one of the best substrates of CaM kinase II in vitro thought to be involved in secretion process. Synapsin I phosphorylation in the neuron by CaM kinase II is essential for neurotransmitter release. Unique to this study, both proteins were determined to be expressed in clonal mouse β-cells (βTC3) and primary rat islet β-cells. By immunoprecipation, in situ phosphorylation of MAP-2 and synapsin I was induced in permeabilized βTC3 cells within a calcium range shown to activate endogenous CaM kinase II under identical conditions. Two-dimensional tryptic phosphopeptide mapping of both proteins revealed that sites phosphorylated by CaM kinase II in vitro, while distinct from sites phosphorylated by protein kinase A in vitro, were largely homologous to those sites phosphorylated in situ upon incubation of the βTC3 cells with increased free calcium. Immunofluorescence verified expression of both proteins in βTC3 cells and pancreatic slices, however, synapsin I exhibited little co-localization with insulin containing dense core granules as demonstrated by immunogold electron microscopy. These data provide evidence that MAP-2 and synapsin I are phosphorylated by CaM kinase II in the pancreatic β-cell in situ. While the data suggest that synapsin I may not be involved in insulin secretion, an association with other known microvesicles of the β-cell, similarly secreted, may be possible. The phosphorylation of these CaM kinase II substrates may reveal an important intermediate step in the mediation of the glucose response in the pancreatic β-cell.