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Biochemistry and molecular Biology.

Monocyte Recruitment, Heterogeneity, and Differentiation in Patients Undergoing Cardiac Catheterization. Amir Al-Dabagh, University of Michigan-Flint, Biology Department

The problem/question of monocyte heterogeneity find recruitment is currently under extensive research and study. This problem is part of the research focused on monocytes and has a great impact on both research and therapies in the medical field. In order to understand the question of monocyte heterogeneity and recruitment, we need to first carefully define some terms including monocyte, heterogeneity, recruitment, mononuclear phagocytes, macrophages, inflammation., atherosclerosis, lymphocytes, hypercholesterolemia, atherothrombosis, and atheromata. We then need to examine the history of the problem of monocyte heterogeneity and recruitment starting in 1968 in order to see how our understanding of the question has developed. Next, we will focus on more recent research on the topic, exploring changes and advances in our perception of the monocyte problem. After looking at recent work, we will focus on the current status of research about the question, and look at future trends and areas of research.

Partial Purification and Characterization of Secreted Proprotein Convertase Furin and PACE4. Arasakumar Subramani, Roxanne M. Casey, and Joseph F. Sucic, Biology Department, University of Michigan--Flint

Subtilisin-like proprotein convertases (SPCs) are a family of mammalian endoproteases. SPCs are responsible for the proteolytic cleavage and subsequent activation of proteins such as clotting factors and other blood proteins, hormones, receptors, growth factors, adhesion molecules, matrix-degrading metalloproteinases, and other enzymes. Moreover, these enzymes have been implicated in many pathological conditions, including Alzheimer's disease, cancer, and a number of infectious diseases caused by both bacteria and viruses. Furin and PACE4 are widely expressed members of SPC family. We examined the substrate specificities of these enzymes. RPE.40 cells, an endoprotease--deffcient strain of Chinese hamster ovary cells, were stably transfected with constructs directing the production of a secreted form of furin and PACE4. Conditioned medium from these cells was collected, and ultrafiltration methods were utilized to partially purity both enzymes. Substrate specificity was examined quantitatively and qualitatively. For quantitative analysis fluorogenic peptide substrates with differing cleavage site sequences were used. Fluorometry was used to quantify reaction products, and the Michaelis-Menten constant, K[sub.m], was ascertained for the different substrates. For qualitative analysis wild type and mutant pro Von Willebrand's Factor were used and the substrate specificity was examined by western blotting.

Effects of Stress Conditions on Subtilisin-like Proprotein Convertase Expression. Krishnapriya Chinnaswamy, Jennifer Ware, Jeremy Lynd, Arasakumar Subramani, Soumya Pal, and Joseph F. Sucic. University of Michigan - Flint, Department of Biology

Subtilisin-like Proprotein Convertases (SPCs) are a family of eukaryotic endoproteases. Cleavage by SPCs activates a variety of proproteins, including hormones, growth factors, clotting factors, matrix-degrading metalloproteinases, receptors, and adhesion molecules. SPCs may be involved in mediating cellular responses to environmental stresses. We examined the expression of the genes for four SPCs (furin, PACE4, PC5, and PC7) under stress conditions. Mouse 3T3 cells were incubated in conditions of heat (42[degrees]C), hypoxia, and both heat and hypoxia. Control cells were incubated under standard cell culture conditions. At three time points (one hour, six hours, and twenty-two hours of incubation), RNA was extracted from the cells under stress conditions; RNA extraction was performed on control cells at only one time point. This RNA was used for cDNA synthesis and quantitative Real-Time PCR to determine if there were differences in expression in stress conditions. Differences in expression were determined by [DELTA]Ct method, which compares the ct values of the SPCs to the Ct values of HPRT, a reference gene used in stress studies. The [DELTA]Ct values were then analyzed using ANOVA to determine if the differences in expression were statistically significant.

A Rapid Test to Identify Northern Milfoil, Eurasian Watermilfoil and the Aggressive Interspecies Hybrid. Nickole Hatley and Ann Sturtevant, University of Michigan-Flint, Biology Department

Watermilfoil is an invasive 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. We have identified genotypes of the native northern milfoil (Myriophyllum sibiricum), the imported plant pest Eurasian watermilfoil (Myriophyllum spicatum) and the aggressive interspecies hybrid (M. spicamm X M. sihiricum) currently present in a number of Michigan lakes. Because identification of watermilfoil species based solely on morphology can be problematic, characterization at the DNA level was done by sequencing the ITS region of the ribosomal RNA genes. However, cloning and sequencing is a lengthy and expensive process when large numbers of samples need to be analyzed. An extremely sensitive molecular technique called real-time PCR (RT-PCR) will be used to develop a diagnostic test to rapidly detect certain genetic markers for the native northern milfoil (M. sibiricum), the imported plant pest Eurasian watermilfoil (Myriophyllum spicatum) and the aggressive interspecies hybrid (M. spicatum X M. sibiricum). These genetic markers will then be used to correlate the presence of specific genotypes of watermilfoil with herbicide tolerance in Lobdell Lake.

Triphenylphosphine Promotes Production of 12-HETE during COX-1 Dependent Metabolism of Arachidonic Acid. Rohini Sidhu, Hemendra Basu, Lalini Ailaboina, Aditi Sengupta, and Steven Pernecky, Department of Chemistry,Eastern Michigan University

Lipid hydroperoxides play a prominent role in reactions catalyzed by cyclooygenase (COX-1). Thin layer chromatography reveals that arachidonic acid (AA) preparations from commercial sources contain lipid hydroperoxides, which can be readily reduced to alcohols by triphenylphosphine (TPP). Prostaglandin [PGE.sub.2] non-enzymatically formed during metabolism by ovine COX--1 from the product [PGH.sub.2], is produced during AA metabolism. When AA pretreated with TPP was incubated with COX-1, which was also treated with TPP, there was a marked decrease in the amount of [PGE sub.2] produced. Production of a novel metabolite, which was identified by GC-MS as 12-HETE, was also observed. When COX-1 was heat-inactivated prior to reacting with TPP-treated AA, no 12-HETE was produced. When the same experiment was conducted with tributylphosphine (TBP), [PGE.sub.2] production was not compromised and no 12-HETE was produced. These results suggest that TPP stimulates a lipoxygenase activity in COX-1 while suppressing cyclooxygenase activity by a mechanism that does not involve the reductive catalytic function.

GC-MS Characterization of Fatty Acids and their Derivatives in Cecal Fluid.

Charles Harrison, Rohini Sidhu, Steven Pernecky

The human gastrointestinal (GI) tract is known to harbor a wide variety of bacterial organisms. Fortunately for humans the majority of the microbiota found in the GI tract are comprised of nonpathogenic anaerobic organisms. The bacterial organisms that produce lactic and butyric acid are known to have positive health benefits. These bacterial products may be used as the preferred energy source for subsequent beneficial organisms, which thus compete with, pathogenic bacteria and yeasts. An analytic assay has been developed that allows for quantitative measurement of these metabolites that are present in the cecal fluid of laboratory mice that are maintained in a germ-free environment, or administered antibiotics. Experimental mice (obtained from Dr. Gary Huffnagle, Univ. of Mich.) are inoculated with, microorganisms found in the human GI tract. The cecal contents are harvested, and ether extraction is performed four times. The biological extracts are then derivatized and prepared for analysis by GC/MS. Metabolic composition in cecal fluid of wild-type mice can then be compared to those of experimental animals that have been inoculated with various types of microorganisms.

Is Soybean More "SLEEPY" than Arabidopsis? Sorting Out Multiple Putative Orthologues of the Arabidopsis Gene SLEEPY-I. Derek Janssens, Grand Valley State University, Department of Biomedical Science

Gibberillic Acid (GA) signaling is critical for multiple developmental processes throughout the life cycle of several plants including Arabidopsis thaliana, barley, grape, maize, rice, and wheat. The F-Box protein Sleepy 1 (SLYI) has been characterized in Arabidopsis thaliana as a positive regulator of GA signaling. The SLYI gene is up regulated in response to an increase in bioactive GA and functions to degrade inhibitors of GA signaling. By screening a Soybean cDNA library, we identified a putative orthologue of the SLYI gene. Interestingly, other studies have found an alternative putative SLYI orthologue in Soybean using bio-informatic techniques. While the protein sequence of both putative orthologues contains an F-Box motif as well as several other domains common to the Arabidopsis SLYI protein, it has not been determined whether these orthologues fulfill the same function as SLYI in Soybean. We treated Soybean, seedlings with either GA or paclobutrazol (an inhibitor of GA biosynthesis); studies in Arabidopsis suggest that the former treatment increases SLYI activity while the latter treatment decreases SLYI activity. Using RT-PCR, we are determining if these treatments have similar affects on the expression of either Soybean putative orthologue.

Characterization of OXSI's Role in Stress Activated MAP Kinase Signaling Pathway in Schizosaccharomyces pombe Using Yeast Two-Hybrid. Allison Barbaglia and Dr. Pei-Lan Tsou, Grand Valley State University, Cell and Molecular Biology

Through a functional screen in the fission yeast Schizosaccharomyces pombe, we uncovered a new gene, oxsl (+), that can enhance tolerance to cadmium or oxidizing chemicals. OXS1 protein accumulates in the nucleus during stress, and nuclear exclusion in the absence of stress requires Crm1 (exportin1). We have identified Oxs1 homologous genes so far in rice, maize, Arabidopsis, fruit flies, mouse and human. It is not only conserved in structure, but it also appears conserved in function. In fission yeast, oxidative stress triggers distinct signaling MAPK pathways. Oxs1 represents a new target for the study of oxidative stress tolerance in eukaryotic organisms. However, Oxs1's enhancement of oxidative stress tolerance is Sty1 dependent. Here, we further characterize Oxs1 and in search of the proteins in the stress pathways that interact with this protein using the yeast-two hybrid system.

Root Cell Shape Is Altered in T-DNA Knockout Eukaryotic Elongation Factor One Alpha Arabidopsis Thaliana Seedlings. Katrina Murphy and Wendy D. Ransom-Hodgkins, Western Michigan University; Department of Biological Sciences

Eukaryotic Elongation Factor One Alpha (eEF1A) GTP binding protein involved in protein synthesis and degradation, binding actin and microtubules, and several different signal transduction, pathways in the cell. The regulation of eEF1A's many functions is not understood. Arabidopsis thaliana has four eEF1A genes forming a small family. We have taken a loss of function approach to gain a better understanding of the importance and functions of each eEF1A family member. To accomplish this task we identified forty T-DNA knockout lines containing a T-DNA insertion in the promoter, coding, or 3'UTR region of each eEFIA gene. Plants were grown under normal growth conditions and the phenotypes were noted. The most dramatic phenotype observed was a change in root morphology. Several T-DNA lines displayed a short root length compared to wild type at six days post germination. Observation of the eEF1A T-DNA seedlings primary root at: twelve days post germination showed a change in cell shape. Wild type root cells were rectangular in shape whereas the eEF1A T-DNA cells were square in the region of maturation. The root phenotype was observed in T-DNA lines spanning the four genes and two different insertion locations.

Localization of Eukaryotic Elongation Factor One Alpha Protein in Arabidopsis thaliana. Stacy Ruszkowski and Wendy D. Ransom-Hodgkins, Western Michigan University; Department of Biological Sciences

Eukaryotic Elongation Factor One Alpha (eEF1A) is a multifunctional protein active in protein synthesis and degradation, binding actin and microtubules and several different signal transduction pathways in the cell. In Arabidopsis thaliana there are four genes for eEF1A: At1g07920, At1g07930, At1g07940 and At5g60390. The specific function of each gene is not known. To study the function of each gene we wanted to visualize each protein's location in the plant. To accomplish this task we cloned the promoter and coding region of each gene and fused to the green fluorescent protein. Agrobacterium mediated transformation was used to create transgenic plants expressing the eEFlA-GFP fusion under its native promoter. Transformed seedlings have been identified and we will describe the localization of the protein throughout development in Arabidopsis thaliana plants.

Insect Mitochondrial Gene Order. Paul Albosta, Seth Eckel, Maria Duclos and David J. Stanton, Saginaw Valley State University, Department of Biology

Mitochondrial gene content and gene order are highly conserved in animals. Indeed, most arthropods share a similar gene order and most crustaceans and insects have an identical gene order. However, some variation in mitochondrial gene order has been detected within certain insect groups such, as Orthoptera, Hemiptera, Lepidoptera and Hymenoptera. Since such variation is rare, rearrangements can serve as valuable markers for phylogenetic reconstruction in groups where they ate found. In order to screen for variation in mitochondria gene order, over 300 insects representing 15 different orders have been collected and total genomic DNA has been extracted from over 100 samples. We have also designed and constructed PCR primers that anneal to conserved regions within insect mitochondrial DNA. These primers allow for the amplification of five gene junctions that collectively contain 17 of the 22 mitochondrial tRNA genes, including junctions known to be hot spots for gene rearrangement events. By screening for size variation in PCR amplifications, we can detect possible gene rearrangement events winch can he confirmed by DNA sequencing. The phylogenetic utility of these variants can then be assessed by screening other members of any group in which variation is detected.
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Publication:Michigan Academician
Article Type:Author abstract
Geographic Code:1U3MI
Date:Dec 22, 2009
Words:2179
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