Biochemistry/molecular Biology.

Mycobacterium chlorophenolicum and Its Ability to Survive in the Presence of Pentachlorophenol. Rachael Glassford, and Darin McCarthy, Calvin College, Department of Chemistry and Biochemistry, Grand Rapids, MI 49546

Pentachlophenol (PCP), a wood preservative banned in 1987 due to its toxicity, continues to contaminate sites around the world and presents an environmental cleanup challenge. M. chlorophenolicum has been isolated from PCP-contaminated sites and has the ability to survive in the presence of PCP, despite the fact that this toxin interferes with oxidative phosphorylation. Out goal is to understand the mechanisms by which M. chlorophenolicum both survives in the presence of, and degrades PCP Although PCP induces a lag phase in M. chlorophenolicum growth, PCP-treated cells grow at the same rate as control cultures. However, PCP inhibits growth in control mycobacteria, M. smegmatis and M. phlei. In addition, cellular respiration in M. chlorophenolicum is significantly inhibited at high concentrations of PCP, but is uncoupled in control strains. The effect of PCP on respiration is pH dependent; oxygen consumption is inhibited more strongly at low pH. These results indicate that M. chlorophenolicum is uniquely able to survive in the presence of PCP because it enters a dormant state at high PCP concentrations, thus not wasting energy that would be lost due to uncoupling of oxidative phosphorylation.

Preliminary Studies on the Dechlorination of Tetrachloro-l,4-hydroquine by Mycobacterium chlorophenolicum PCP-1. Arianne Folkema, Brad Veldkamp, Lia Wisniewski, and Darla McCarthy, Calvin College, Department of Chemistry and Biochemistry, Grand Rapids, MI 49546

We are characterizing a tetrachloro-1,4-hydroquinone (TCHQ) dehalogenating enzyme from Mycobacterium chlorophenolicum PCP-1, a gram-positive bacterium that catabolizes pentachlorophenol (PCP). The PCP catabolic. pathway in M. chlorophenolicum PCP-1 begins with the para-hydroxlyation of PCP to form TCHQ. The next step in the catabolic pathway, catalyzed by TCHQ dehalogenase, has been proposed to involve hydroxylation of TCHQ to form tnchloro-l,2,4-trihydtoxybenzene (TTHB). However, our preliminary results indicate that a reductive dehalogenation occurs, producing trichloro-l,4-hydroquinone. Such a reductive dehalogenation is known to be catalyzed by the TCHQ dehalogenase isolated from Sphingobium chlorophenolicum. The reducing equivalents for the reductive dehalogenation catalyzed by the S. chlorophenolicum TCHQ dehalogenase come from the co-substrate glutathione. However, mycobacteria are known NOT to synthesize, or even to utilize, glutathione. Indeed, out results indicate that the activity of TCHQ dehalogenase from M. chlorophenolicum PCP-1 is not dependent on glutathione. We are currently attempting to determine the identity of the reducing co-substrate employed by the M. chlorophenolicum PCP-1 TCHQ dehalogenase.

Identification of Host and Bacterial Proteins Present Inside Murine Abscesses Infected by Staphylococcus aureus. Marcella Luercio, University of Michigan-Flint, Biology Department, Flint, MI 48502, Eric Skaar and Victor Torres, Vanderbilt University, Department of Microbiology and Immunology

Staphylococcus aureus is a prominent gram-positive human pathogen, causing injections worldwide. The isolation of S. aureus strains that ate resistant to multiple antibiotics and the ability of this pathogen to evade the host's immune response emphasizes the importance of understanding the virulence mechanisms of S. aureus and bacteria-host interaction at the molecular level. Bacterial and host proteinspresent inside murine abscesses infected with S. aureus were identified using a combination of Imaging Mass Spectrometry (IMS) and Shotgun Proteomics. Five genes, encoding the identified proteins, were deleted from the bacterial genome through allelic replacement. These genes may encode proteins that are involved in virulence mechanisms. The introduction of the mutants into a mouse model may provide insights into the identification of a large number of proteins whose functions ate currently unknown. This will lead to the assigning of functions to these proteins (in relation to pathogenesis) and providing a more clear understanding of the factors involved in S. aureus' virulence mechanisms and avoidance of the immune response.

The Influence of Antibiotics on Bacterial Motility and its Implication for Drug Efficacy in Micro gravity. Anna Stanczyk, University of Michigan-Flint

Understanding in vitro bacterial response to antibiotics in a microgravity environment is an important step toward the goal of minimizing astronaut health risks. Previous studies have shown that bacteria cultured in space are able to proliferate in normally inhibitory concentrations of antibiotics. Identifying the cause of this reported behavior has proven difficult, however, due to the complex interactions of gravitational influence on living organisms. Research aimed at identifying and isolating independent variables can help to elucidate the responsible mechanisms. This project focuses on the study of one such variable, cell motility. It is hypothesized that antibiotic effectiveness in space is reduced as an indirect consequence of bacterial motility inhibition. The rationale behind our hypothesis is based on related research suggesting that less motile bacteria exhibit more pronounced responses to microgravity in general than highly motile ones, purportedly due to extracellular mass transport factors. Motility of E. coli (ATCC 4157) was evaluated as a function of radial growth from a stab culture inoculated on semi-solid agar using different nutrient sources, both with and without Streptomycin. Results are correlated to literature describing antibiotic experiments conducted in space.

Production of AcpA- and AcpA-/AcpC- Francisella novicida Mutants and Characterization Using Fluorescence Activity Assay and Reverse Transcriptase PCR. Brendan Yonke, University of Michigan-Flint

Francisella novicida was used as a model for the intracellular bacteria F. tularensis, the causative agent of the tularemia. Previously, acid phosphatases of intracellular bacteria have been shown to inhibit respiratory bursts in neutrophils, possibly indicating the role of such enzymes in F. novicida intracellular survival. Analysis of the F. novicida genome has identified the presence of multiple acid phosphatase encoding genes including AcpA and AcpC. As a first step towards determining the role of acid phosphatases in intracellular survival, AcpA- and AcpA-/AcpC- F. novicida strains were created using pre-existing AcpC- and wild type strains. In this research, performed under Dr. Gunn at the Ohio State Medical Center, cryotransformation and subsequent homologous recombination events involving a AAcpA plasmid were used to generate AcpA- and AcpA-/AcpC- mutants. Following confirmation of mutations, qualitative analysis of the generated strains was performed using fluorescent acid phosphatase activity assays and reverse transcriptase PCR. Comparison of the generated strains to existing AcpC- and wild type strains served to determine the effects of AcpA- and AcpA-/C- genotypes upon acid phosphatase activity and expression in F. novicida.

The Effect of Vision Loss on Subtilisin-like Proprotein Convertase Expression in the Brain. Andrea MacFadden, Biology, Michael Jarvinen, Psychology, and Joe Sucic, Biology, University of Michigan--Flint, Flint, MI 48502

Subtilisin-like proprotein convertases (SPCs) arc capable of processing a number of proproteins in the secretory pathway known to affect nervous system development and synaptogenesis. Currently, very little is known about the expression of SPCs in the diseased brain. To address this question, we used a murine model of retinal degeneration (Pde6b- mice) that leads to progressive vision loss between postnatal days (PNDs) 95-113 (N=66). We sampled brain regions (visual cortex, olfactory cortex, and cerebellum) from Pde6b- mice before and after PND 95-113 and processed this tissue using commercially available RNA isolation and cDNA synthesis kits (N=ll). RT-PCR analyses included sequence-specific SPC primer sets (PC1, PC2, PC4, PC5A, PC5B, PC7, Pskn9, PACE4, and furin) and the constitutively expressed gene for GAPDH (used as an internal standard for semi-quantitative analysis). Distinct patterns of SPC gene expression were detected. For instance, furin levels decreased substantially in the visual cortex after blindness ensued whereas PACE 4 levels remained stable. Analyses of corresponding tissue from control animals (with sight) demonstrated that alterations in SPC expression could not be explained by normal developmental gradients. These results identify changes in SPC gene expression resulting from blindness and provide compelling rationale to conduct further protein analysis studies.

Investigation of Adaptor Molecules' Interactions with Toll-like Receptors (TLR) of the Mammalian Cornea: Roles of TRIF and MyD88 in TLR3 Expression and Mediated-Immune Response. Safa Al-Dabagh, University of Michigan-Flint, Department of Biology; Eric Pearlman, Case Western Reserve University, Department of Ophthalmology

Toll-like receptors (TLRs) play a critical role in innate immune responses through the recognition of a variety of microbial products. While many TLRs signal through MyD88, TLR3 and a subset of TLR4 activation events are known to signal through a MyD88-independent pathway, involving the adaptor molecule TRIF [TIR domain-containing adaptor inducing IFN-[beta]]. To this end, current studies have endeavored to determine the presence and possible role of a MyD88- independent pathway of corneal inflammation. Thus, in vivo mouse experiments were performed demonstrating the exacerbation of Poly(I:C) induced inflammation in MyD88 knockout mice. Human Corenal Epithelial-T cells studies further confirmed that inflammatory responses are increased when MyD88 expression is deficient. These findings are consistent with a regulatory role for MyD88 on the TLR3/TRIF pathway, a link that has not been previously observed. This research, attempting to study roles of adaptor proteins, TLR3 and MyD88, is important in providing a deeper understanding of TLR3's role in corneal inflammation, allowing for better designs in more effective anti-viral therapies.

Evolutionary History of ID Elements in the Rodent Suborder Hystricognathi. Lindsey S. Beitler and David H. Kass, Eastern Michigan University, Department of Biology, Ypsilanti, MI 48197

ID elements are a family of short insterspersed DNA elements (SINEs) derived by retrotransposition from the BCl master gene. Orders of magnitude variations in copy number have been observed among the genomes of myomorphic rodents. In contrast, only very modest levels have been demonstrated in nutria and guinea pig, that are both classified in. the Hystricognathi suborder of rodents. By intra-ID PCR and dot blot analyses, we are determining the comparative amplification success of ID elements in the hystrieognathid rodents to further elucidate their evolutionary history. Surprisingly, distinct, larger bands have been detected in the nutria and degu using intra-ID PCR. We are currently investigating the possibility of uncharacterized ID-like subfamilies within these rodent species. Additionally, we are using AB-PCR and A-PCR technique to identify retrotransposons within the rabbit. This work will provide new insights regarding the evolutionary history of ID elements, as well as potentially offer new tools for phylogcnctic analyses.

Identification of a New Family of Short Interspersed DNA Elements within the Guinea Pig (Cavia porcellus) Genome. Brian A. Schaetz, Kevin Bonney, and David H. Kass, Eastern Michigan University, Department of Biology, Ypsilanti, MI 48197

Short interspersed DNA elements (SINEs) constitute large proportions of human and mouse genomes. Theses elements accumulated in these genomes by the process of retrotransposition. Known rodent SINEs such as Bl, B2 and ID elements are abundant in mice and rats, but are either present in low copies or absent within the guinea pig genome. To gain further insight into the paucity of SINEs within the guinea pig, we utilized the techniques of A-PCR and AB-PCR to potentially identify undocumented SINEs. We have uncovered a previously uncharacterized, ID-like SINE family in the guinea pig, and have determined its presence within other rodents of the hystricognathid suborder. We will continue to determine copy numbers and analyze sequences to further elucidate the evolutionary history if this SINE family.

Assessment of Germ-Line Transcription of Retrotransposons in Rodents. Nicole Jamison and David Kass, Eastern Michigan University, Department of Biology, Ypsilanti, MI 48197

Retrottanspons are a group of transposable elements whereby new copies integrate into the genome via an RNA intermediate. Guinea pig genomes contain a relative paucity of known rodent retrotransposons in relation to the mouse and rat. In this investigation we utilize the techniques of RT-PCR and C-RACE in order to identify transcripts in germ-line cells in rodents, as well as to potentially identify the loci responsible for generating new elements. By analyzing products using agarose gel electrophoresis, we observed similar banding patterns among tissues for all rodents for the Bl retrotransposon. However, the B2 element band present in mouse and hamster was lacking in the guinea pig. This is consistent with the lack (or possible absence) of B2 elements within the guinea pig genome. Additionally, we have thus far identified transcripts exclusively of the "young" Bl subfamilies in the mouse, which would be expected, as we do not anticipate the older elements to be active. Also, we have isolated transcripts of the BC1-1 and BCl-2 guinea pig master genes for ID elements in the testes and ovaries, respectively, possibly suggesting tissue-specific expression. Our inklings also suggest that genes other than BCl serve to generate new ID elements.

Retrotransposition of the Hamster B2 Short Interspersed DNA Element. Joseph A. Katakowski, Erin Radloff, Eillen Tecle, and David H. Kass, Eastern Michigan University, Department of Biology, Ypsilanti, MI

Short interspersed DNA elements (SINEs) represent a family of transposable elements (TEs) within mammalian genomes. The rodent B2 SINE family has amplified to approximately 80,000 copies among myomorphic rodents. The B2 element displays a high level of sequence similarity between hamster and mouse, with the exception of the .3' end. We have identified recent B2 element integrations in the genomes of various species of hamster. This provides a useful phylogenetic tool, which further supports a recent study that greatly restructured the hamster taxonomy. We are also incorporating a presumptive active hamster B2 element into a construct that will allow for the assessment of its "jumping" ability using a cell culture assay. We have determined that the mouse B2 element appears to undergo retrotransposition equally well when it utilizes either a human LINE or a mouse LINE. This study will determine the relative efficiency of the hamster B2 element to "jump" using the mouse and human LINE. Overall, this work will provide insights into the mechanisms involved in the generation of TEs in mammalian genomes as well as depict recent activity of B2 elements within hamster genomes.

Molecular Characterization of Watermilfoil, an Invasive Aquatic Plant, in Michigan Lakes. Nickole Hatley, Ryan Sutter, Ryan Sheick, Genevieve Colon, Alan Mortimer, David Shorez and Ann Sturtevant, University of Michigan-Flint, Biology Department, Flint, MI 48502

Watermilfoil is an invasive, noxious aquatic plant that seriously impacts the water recreation and tourism industry of Michigan. The introduced, invasive form of watermilfoil can be devastating to aquatic ecosystems where it replaces native vegetation and negatively impacts species diversity among both plants and animals. Watermilfoil is difficult to control because of its rapid growth. We have identified genotypes of the native northern milfoil (Myrtophyllum sibiricum), the imported plant pest Eurasian watermilfoil (Myriophyllum spicatum) and the aggressive interspecies hybrid (M. spicatum X M. sibiricum) currently present in a number of Michigan lakes. Identification of watermilfoil species is done by sequencing the ITS region of the ribosomal RNA genes. An extremely sensitive form of DNA fingerprinting, AFLP, will be used to characterize specific genotypes of each species of watermilfoil. There have also been reports of. herbicide-resistant forms of watermilfoil in Michigan. The genetic markers produced by AFLP will be used to diagnose the presence of herbicide-resistant varieties of watermilfoil and determine its prevalence in Michigan lakes. These genetic markers can be used in the future to identify which lakes have the most aggressive form of watermilfoil, and to recommend the most appropriate herbicide to control this invasive aquatic plant.

Arachidonate Oxidation Metabolite Profiles for Myoglobin and a Structurally Altered Form of Myoglobin Produced in Muscle Disease or Trauma. Kathryn Lawrence, Lalini Ailaboina, Vasumathi Molugu, Steven Pernecky, and Hemendra Basu, Eastern Michigan University, Department of Chemistry, Ypsilanti, MI 48197

Myoglobin (Mb) is released to the blood from diseased or traumatized muscle, and promotes oxidation of lipid membranes of the kidney that may lead to damage, although the extent to which myoglobin and an oxidatively modified form of myoglobin in which the heme is covalently bound to the protein known as myoglobin-H (MbH) contribute to the kidney damage is not known. In a previous study, phoshatidylcholine (PAPC) containing arachidonic acid, a twenty-carbon polyunsaturated tarry acid, was incubated with Mb or with MbH in 0.1 M sodium acetate, pH 6.5. Several aldehyde breakdown products of arachidonic acid, including a three-carbon malondialdehyde and the nine-carbon 4hydroxynonenal were produced during oxidation with Mb or MbH, although Mb generates greater quantities of these products than does MbH. The focus of this research of the present study was to determine whether Mb or MbH produce prostaglandin or hydroxyl-containing oxidation products of arachidonic acid. A combination of thin layer chromatography, ion-trapping gas chromarography-mas'-spectrometry, high pressure liquid chromatography, and quadrapole mass spectrometry was- used to characterize these oxidarion products, and the results reveal that a metabolite is produced coincident with MbH oxidation of PAPC, but not when Mb is used as the pro-oxidant.

Mass Spectrometric Determination of Glycation Sites in Hemoglobins. Billy Clifford-Nunn, University of Michigan--Flint, and Sonja Hess, National Institutes of Health, National Institutes of Diabetes and Digestive and Kidney Diseases

Much information is known about hemoglobin and sickle cell anemia, however, treatments are limited and diagnostic tests problematic. Because of these problems, researchers are exploring new treatment and diagnostic methods, Electrospray ionization mass spectrometry (ESI-MS) consistently detects the differences in hemoglobin variants with precision and accuracy.

Research being carried our at the National Institutes of Health using this technology may be the key to determining new diagnostic tests and treatments. The current paper focuses on this research. Fetal hemoglobin standards, sickle cell hemoglobin standards and hemoglobin variants were isolated from, diabetic patients. The alpha, beta, sickle, gamma and glycated chains were identified using liquid chromatography mass spectroscopy (LC-MS). The main peaks on the chromatograms produced from these experiments were assigned as the separate subunits because of their molecular weights. Each fraction was digested using trypsin, an enzyme that breaks bonds between certain amino acids. The peptides were then sequenced using LC-MS/MS. To verify which peptides were known, database searches were performed. It was also from these searches that the appropriate peaks were labeled with the appropriate subunit.