Hood College Biomedical and Environmental Science
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Item PCR AMPLIFICATION AND CLONING OF ELEVEN OVERLAPPING SEGMENTS OF BIV127 env FOR BACTERIAL EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF EXPRESSED PROTEINS(1995-11) Hutchison, Don C.; Hood College Biology; Biomedical and Environmental ScienceBacterially expressed segments of bovine immunodeficiency virus (BIV) envelope were tested for immunological reactivity with sera from cows, rabbits, and a guinea pig. Overlapping segments of the gene coding for BIV127 envelope surface (SU) and transmembrane (TM) regions were generated by the polymerase chain reaction and subcloned into two fusion protein expression systems: pGEX 4-T-1, a glutathione-Stransferase (GST) fusion system (Pharmacia), and pMAL-C2, a maltose binding protein (MBP) fusion system (New England Biolabs). E. co//transformed with the recombinant plasmids were induced to express BIV-GST or BIV-MBP fusion proteins. The fusion proteins were purified by affinity chromatography using Glutathione Sepharose 4B or amylose resin, respectively. The BIV-MBP peptides were then separated by size on SDS-PAGE gels and transferred onto lmmobilon filters by semi-dry blotting. BIV-MBP purified fusion proteins were also cleaved by factor Xa protease to release the BIV fragment from its fusion partner prior to separation by SOS-PAGE gradient gels and western blot. Fusion proteins were detected using goat anti-GST antibodies or rabbit anti-MBP and tested for reactivity with sera from naturally and experimentally infected cows, an experimentally infected rabbit, immunized rabbits, and an immunized guinea pig. Additionally, sera to synthetic peptides derived from the BIV env gene were tested. Primary sera were incubated with an appropriate horseradish peroxidase-conjugated (HRP) secondary antibody using the enhanced chemi-luminescence system and exposed to x-ray film. The pGEX (GST) expression system showed low yields of BIV-GST fusion protein compared to GST controls expressed with no fusion partner. In contrast, relatively large amounts of BIV-MBP fusion proteins were expressed and could be visualized by Coomassie staining on SDS-PAGE gels as well as western blot. Analysis of the SDS-PAGE and western blot results (of BIV-MBP antigen against cow and rabbit sera) showed BIV reactivity in the BIV-MBP fusion proteins. However, with some antigens, normal control sera also reacted to the BIV-MBP fusion proteins or to copurified protein(s) of similar molecular weight. To evade "non-specific" MBP or copurified protein reactivity, BIV-MBP fusion proteins were cleaved with factor Xa and electrophoresed on 4-20% SDS-PAGE gradient gels prior to western blotting, and tested with a similar panel of sera. An immunogenic region of the amino-terminus of TM corresponding to the extracellular domain (represented by TM1-MBP) was recognized by sera from a naturally infected cow and a cow transfused with a field isolate in both the uncleaved and factor Xa cleaved experiments. Rabbit sera from a BIV127-infected rabbit and a rabbit immunized with purified BIV virion also recognized the cleavage product from factor Xa cleaved TM1-MBP antigen.Item KINSHIP AND AGONISTIC BEHAVIOR AMONG MALE MICE(1995-05) Hughes, Melanie K.; Hood College Biology; Biomedical and Environmental ScienceAdult male mice defend small territories and compete for mates by engaging in agonistic interactions. A dominance hierarchy is established based on the outcome of these agonistic encounters and the urinary marking behavior of adult male mice is usually strongly dependent on their social dominance ranks. Animals do not behave selfishly all the time but in some circumstances may behave cooperatively, particularly with kin. Hamilton (1964) proposed the theory of kin selection which predicts that the evolution of social behavior is influenced by the degree of kinship between two individuals. I tested Hamilton's theory, that kinship influences agonistic behavior in a preliminary experiment in which I observed agonistic encounters between brothers and nonbrothers. If kin selection on male mice influences interactions between brothers, agonistic interactions between them should be infrequent relative to nonbrothers. The preliminary experiment showed that the mean latency to fight was longer for brothers than for nonbrothers. The mechanism(s) controlling this nepotistic behavior among brothers may be emotional attachment due to association of littermates during early development and/or degree of relatedness per se. I used a cross-fostering procedure to establish four categories of male mice: brothers in same litter, nonbrothers in different litters, brothers in different litters, and nonbrothers in same litter. My results indicate that both attachment and relatedness influence agonistic interactions. To investigate the relevance of open arena tests and whether male mice will continue to display nepotistic behavior in the presense of a limited resource, the pairs of mice were fought a second time with an estrous female present. The male mice had significantly lower latencies which may have resulted from their prior fighting experience, although the pattern of latencies in relation to attachment and relatedness remained the same. Finally, I tested whether kinship influences the nature of urinary marking patterns of male mice and found no significant difference in the urine pattern.Item THE BACTERIAL EXPRESSION AND CHARACTERIZATION OF BIV REV EXON 2 AND THE EXTRACELLULAR DOMAIN OF BIV TRANSMEMBRANE PROTEIN(1996-04) Hu, Marie Y.; Hood College Biology; Biomedical and Environmental ScienceTo determine if BIV rev exon 2 is post-translationally removed from the Env polypeptide and if the extracellular domain of BIV TM protein is the region responsible for virus-cell fusion, PCR generated fragments of these two regions were cloned and bacterially expressed as maltose binding protein (MBP) fusion proteins in the pMAL expression system. The expressed fusion proteins were isolated and purified by affinity chromatography using amylose resin. The fusion proteins were immunologically characterized by immunoblotting using BIV specific sera, as well as the rabbit anti-MBP serum. The purified proteins were used to immunize mice and guinea pigs for the production of antisera. The antisera were immunologically characterized by western blotting and radioimmunoprecipitation of BIV 127-infected cell lysates. The experimental findings suggest that rev exon 2 translation appears in both the Env precursor and the mature Rev protein. Furthermore, rev exon 2 is posttranslationally cleaved from the gp100 Env surface glycoprotein as evidenced by the recognition of the mouse antisera to MBP-rev exon 2 to p102 Env precursor and the p23rev but not gp100. The presence of the putative BIV primary fusion domain within the extracellular domain of the BIV transmembrane protein (TM-ecd) was not determined due to the lack of reagents.Item IgA1 PROTEASES(1986-08) Hsu, S. Dana; Hood College Biology; Biomedical and Environmental ScienceIgA1 protease is elaborated by several mucosal pathogens, including Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, as well as Streptococcus sanguis, a predominant bacterium in dental plaque. The enzyme is specific for the IgA1 subclass, cleaving the molecule at the hinge region to form intact F ab and F c fragments. IgA2 is not susceptible to proteolysis because of a deletion of a 13 amino acid segment in the hinge region where the cleavage sites are located. S. sanguis 10556 was grown in carbon excess and carbon limitation, in undialyzed and diafiltrated media, and the enzyme partially purified by gel filtration and anion exchange chromatography using high performance liquid chromatography (HPLC). Comparison was made of the elution profiles of the enzyme preparations in gel filtration using phosphate buffer, pH 7.0, and Tris(hydroxymethyl)amino methane-HCL (Tris-HCL) buffer, pH 8.0. IgA1 protease was found to be elaborated under carbon limitation, a condition encountered by microorganisms in dental plaque. The use of diafiltrated medium reduced significantly a large contaminating peak which elutes in approximately the same region as the enzyme in gel filtration (phosphate buffer). Better separation of the enzyme from other components eluting at the void volumn was obtained using phosphate buffer than Tris-HCL buffer in gel filtration. In anionic exchange chromatography, the enzyme eluted unbound, while the majority of the contaminating proteins remained bound to the support. This procedure is an efficient method of obtaining a preparation of S. sanguis IgA1 protease which is contaminated by only a few proteins.Item RESPONSES OF Nf1Fcr/Nf1FcrB CELLS AND Nf1+/Nf1+B CELLS TO SURFACE IMMUNOGLOBULIN CROSSLINKING(1996-11) Householder, Deborah Barnhart; Hood College Biology; Biomedical and Environmental ScienceThe aim of this research is to compare the cellular responses of neurofibromin deficient (Nf1-/-) B cells and neurofibromin positive (Nf1+/+) B cells to surface immunoglobulin (sIg) crosslinking, which mimics B cell activation by specific antigens. Mice heterozygous for a germline null mutation at the Nf1 gene (Nf1Fcr/+) were mated, and fetal liver cells from 13.5 day Nf1Fcr/Nf1Fcr or Nf1+/Nf1+ embryos were used to reconstitute the hematopoietic system of lethally irradiated recipient mice. After full reconstitution had taken place, splenic B cells were isolated and surface immunoglobulin was crosslinked with goat a-murine Ig antibodies. The rate of surface Ig patching and capping after crosslinking was identical in B cells of Nf1Fcr/Nf1Fcr and Nf1+/Nf1+ genotypes. 3H-Thymidine uptake, after crosslinking with α-IgG or α-IgM or with the addition of IL-5, was measured and found to be the same in B cells of both the Nf1Fcr/Nf1Fcr and Nf1+/Nf1+ genotypes. Furthermore, both Nf1 mutant and wild-type B cells exhibited the ability for Ras to cocap with slg. However, differences in the appearance of proteins phosphorylated on tyrosine after crosslinking of surface Ig were noted. When lysates from crosslinked B cells were probed with α-phosphotyrosine antibody a band of approximately 109 kd, present in wild-type lysate, was not present in the mutant lysate. There was also a difference between mutant and wildtype B cell lysates in the level of phosphorylation on tyrosine for other substrates. These results show that neurofibromin may not have a role in the initial activation of the signaling pathway in B cells, but it may play a role further downstream in the signaling pathway. While defects in B cells have not been noted in NF1 patients, who carry one mutant NF1 allele, these results may lead to new insights in neurofibromin function or help diagnose NF1 syndrome in some patients.Item Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Antibodies to Epstein-Barr Virus (EBV)-Associated Viral Capsid Antigen (VCA), Early Antigen (EA), and Nuclear Antigen (EBNA)(1982-08) Hopkins, Ralph F. III; Hood College Biology; Biomedical and Environmental ScienceDevelopment of an enzyme-linked immunosorbent assay (ELISA) is described for the detection of antibodies to Epstein-Barr virus (EBV)-associated proteins including viral capsid antigen (VCA), early antigen (EA), and nuclear antigen (EBNA). The specificity of the three ELISA systems was demonstrated by the use of 43 well-characterized human sera shown by immunofluorescence (IF) to be variously reactive for antibodies to the viral antigens. Of the cell lines tested as sources of ELISA antigen, the productively EBV-infected cell line, B95-8 was selected as the source of VCA and EA while the EBV-genome positive non-virus producer cell line, B1-19 was chosen as a source of EBNA. Nine sera which were negative for antibodies to VCA, EA, and EBNA by IF were also negative by ELISA. Approximately 50% of the positive antisera tested gave higher anti-VCA and anti-EA titers by ELISA than by IF. The EBNA ELISA test was also more sensitive than IF with all 33 EBNA positive sera. The antibody titers obtained for either VCA, EA, or EBNA when measured by IF and ELISA showed a high degree of correlation whereas no correlation was observed between the antibody being detected in the VCA and EA ELISAs. The antibody titers obtained in each of the ELISAs were reproducible within one two-fold dilution between repeated experiments. These ELISAs for EBV-associated antigens are specific, sensitive, rapid, and objective and thus provide a valuable method for detection of antibodies to these antigens.Item Ultraviolet Carcinogenesis in C₃H Athymic Nude Mice(1985-05) Hoover, Tammy; Hood College Biology; Biomedical and Environmental ScienceThe induction of skin tumors by ultraviolet irradiation (UV) in C₃H mice and congenic C₃H nude mice was examined. Prior to UV-irradiation, C₃H nude mice were reconstituted with thymus grafts from C₃H mice. C₃H nude skin was grafted onto the backs of C₃H mice before UV treatment. UV-induced tumors were tested for antigenicity and were examined histologically. The probability of tumor development was similar for UV-irradiated C₃H nude mice, UV-irradiated C₃H nude mice reconstituted with thymus grafts and UV-irradiated C₃H mice grafted with C₃H nude skin. UV-irradiated C₃H mice had a longer latency period of tumor development than the other three treatment groups. The probability of tumor development was significantly (P < 0.0048) lower for the UV-irradiated C₃H mice when compared to the other UV-irradiated treatment groups. All UV-induced tumors in the study were squamous cell carcinomas. Tumors from all of the UV-irradiated treatment groups were antigenic. This study indicates that the differences in latency period and probability of tumor incidence in the nude mouse are due to intrinsic properties of the skin and not to the lack of a functioning thymus.Item Genetic Variation of Neutral and Non-Neutral Markers of the Florida Manatee (Trichechus manatus latirostris)(2013-05) Hogan, Priscilla; Hood College Biology; Biomedical and Environmental ScienceThe Florida manatee, Trichechus manatus latirostris, faces challenges from environmental threats and human interactions. The basis for this project was to characterize and assess the genetic diversity of a neutral marker in the mitochondrial DNA (mtDNA) and a non-neutral marker, a major histocompatibility complex (MHC) gene, of the southwest Florida manatee. There are currently no published data available for the genetic sequences of MHC genes in the Florida manatee. Understanding the genetic variation of MHC genes is important because it may provide insight into the manatees' susceptibility to pathogens and environmental toxins that afflict natural populations. Characterizing a neutral marker of the mtDNA will assess the overall diversity of the population. An analysis of the mtDNA revealed that there was no genetic diversity between the samples characterized. Understanding the genetic differences of both neutral and adaptive markers between individuals in the population is important for the protection of the Florida manatee and the conservation of their habitat and biological corridors.Item NEW REQUIREMENTS FOR PRESCRIPTION DRUG LABELING: A PRELIMINARY EVALUATION OF EFFECTIVENESS(2008-05) Hoffman, Nicole; Hood College Biology; Biomedical and Environmental ScienceMedication errors and their role in patient safety have recently become a widely publicized issue in public health. In an attempt to lower medication errors, the FDA implemented new requirements for prescription drug labeling in June 2006. The new regulations modify the format and content requirements for prescription drug labeling, or package inserts, used by prescribing professionals. The main purposes of the new format are to make it easier for physicians to use the package insert and to make it easier for them to find specific information at the time of prescribing. A package insert that is easier to use will be referenced more frequently, which should lead to fewer medication errors. This study utilized 12 volunteers to evaluate a total of 50 package inserts, in both the old and new format, to see if the new format is easier to use than the old format. The results of the study show that the new format is a statistically significant improvement upon the old format for all evaluated characteristics. However, while the new prescription drug labeling requirements will likely reduce medication errors, public health is a complex issue and an improvement in prescription drug labeling is only one step toward a larger goal of protecting patient safety.Item THE SEQUENCE, GENOMIC ORGANIZATION, AND EXPRESSION OF THE SMALL GENOMIC SEGMENT OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS FOR THE POTENTIAL USE AS A DIAGNOSTIC ANTIGEN(1992-04) Hodgson, Loreen; Hood College Biology; Biomedical and Environmental ScienceCrimean-Congo hemorrhagic fever (CCHF) virus (Nairovirus, Bunyaviridae) is the causative agent of a serious, often fatal human disease. This disease is present in at least 28 countries and may occur even more widely within the range of its Hyalomma tick vector in Africa and Eurasia. In addition to the naturally acquired disease, the virus is also noted for its ability to cause nosocomial outbreaks, as has occurred in at least seven countries in Asia, the Middle East and Africa. These incidents have resulted in mortality rates as high as 80% among infected health care workers. Current laboratory tests for diagnosis and surveillance of CCHF, lack sensitivity and do not differentiate adequately between the human pathogenic and nonpathogenic members of the Nairovirus genus. The disease syndrome in man is well-described clinically, however, the biochemical properties of the virus are poorly understood. Therefore, a molecular and antigenic analyses of the pathogenic CCHF virus was initiated to define the genes which are relevant to viral diagnosis and immunoprophylaxis. Previous monoclonal antibody studies have indicated that the nucleocapsid (NC) protein was the most type-specific polypeptide. Therefore, subsequent studies to be described, have focused on the determination of the sequence of the CCHF virus S RNA segment. In other Bunyaviridae viruses, the S RNA segment of the viral genome encodes the nucleocapsid (NC) protein. This sequence information will contribute towards the development of polymerase chain reaction (PCR) diagnostic assays, and expression of the NC protein for use as a type-specific diagnostic antigen. An efficient in vitro system to replicate CCHF virus was developed using low multiplicity of infection (MOI) of SW13 cell cultures. Comparative studies of the viral proteins of CCHF virus and other Nairoviruses were initiated using this system. These studies demonstrated a common pattern of polypeptides, consisting of two glycoproteins with relative molecular weights (Mr) of 78 and 37 kDa, and a NC polypeptide with 53.9 kDa Mr, comprising the major structural proteins were present among all the Nairovirus that were evaluated. A pulse-chase experiment indicated that an additional three polypeptides are present in CCHF virus-infected cells, which may be precursors in the formation of the virion glycoproteins. RNA was extracted from viral nucleocapsids purified from infected cells by equilibrium centrifugation, and the L, M, and S RNA segments were separated by gel electrophoresis. Based on the 3' end sequence of the S RNA segment, a single S RNA-specific primer was designed to initiate first strand cDNA synthesis and also to initiate synthesis of a full-length PCR product using CCHF virus RNA as a template. The initial PCR product was found to be specific for the CCHF virus S RNA segment when used as a hybridization probe against CCHF virus and other Nairovirus RNAs in a northern blot analysis. This full-length PCR product was modified for use as a sequencing template and both strands of the DNA product were sequenced using T4 DNA polymerase and in a dideoxy sequencing reaction. The CCHF virus S RNA segment was found to be 1672 nucleotides in length. Computer analysis of this sequence predicted a single open reading frame (ORE'), which could encode a 482 amino acid (53.9 kDa) protein. A PCR product representing this ORF, with exogenous terminal restriction endonuclease sites, was inserted into Autoqrapha californica nuclear polyhedrosis virus (AcNPV), a baculovirus, in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera fruqiperda (Sf9) cells were infected with the recombinant baculovirus and monitored for NC protein expression. The recombinant expressed protein was characterized using immunofluorescent (IFA) and immunoprecipitation (IP) assays and was found to be antigenically indistinguishable from the authentic CCHF virus NC protein.Item THE CELL CYCLE EFFECTS AND INDUCTION OF APOPTOSIS IN CCRF-CEM CELLS TREATED WITH NEW COMPOUNDS(1995-07) Hite, Karen M.; Hood College Biology; Biomedical and Environmental ScienceThe National Cancer Institute (NCI) has been investigating anticancer drugs since its inception. A drug screening program has been part of this drug discovery strategy since 1955. The goal of these efforts has been to identify new candidate drugs and toward clinical efficacy. In the present study, a set identified as having NCI Primary In Vitro for their ability to interesting direct their development of four new lead compounds compound profiles in the Cancer Screen were studied in detail induce cell cycle arrest and induction of apoptosis in a human acute lymphoblastic leukemia cell line, CCRF-CEM. Two of the compounds show a COMPARE pattern typical of known tubulin binding compounds and the other two compounds show striking antileukemia activity. The compound effects were analyzed by flow cytometry for their cell cycle activity. The flow cytometry methodology was first calibrated using a set of five well characterized standard compounds. Standard compounds affecting the cell cycle at the S and G2+M phase and resulting in a reduction of the number of cells in the Gi phase with an accumulation of cells in G2 are represented by vinblastine, ncocdazole and taxol. Hydroxyurea and actinomycin D also affect the cell cycle in S phase, but these compounds block the cells in G1 and S and reduce the number of cells in G2. Flow cytometry analysis indicate that the compounds producing differential anti-leukemia activity have no cell cycle effects, while the putative tubulin inhibitors induced cell cycle arrest resulting in an enrichment of cells in G2. These compounds were also analyzed for their ability to induce apoptosis, by measurement of non-isotypic DNA end extension in situ, CCRF-CEM was shown to express the cell surface apoptosis antigen Fas/Apo-1. The set of compounds characterized by antileukemia activity in the primary screen induced apoptosis in CCRF-CEM cells quantitatively equal to the positive control. The second set of compounds failed to induce apoptosis at any concentration tested.Item Determination of Antibiotic Resistance in Bacillus Isolated from Soil(1981-05) Hill, Valerie L.; Hood College Biology; Biomedical and Environmental ScienceIn order to determine antibiotic resistance in Bacillus, Gram positive bacilli were isolated from soil collected from a number of different sites in Frederick County, Md. The Bacillus strains were then tested by the standard agar dilution method for determining antibiotic resistance. Several strains which showed apparently high levels of resistance by this method were subsequently tested quantitatively. The titration of the number of surviving cells against increasing concentrations of antibiotic did not correlate with the Minimal Inhibitory Concentration (NI) determined by the agar dilution method. There was in fact no significant degree of drug resistance demonstrated in any strain of Bacillus isolated.Item K-ras ACTIVATION IN RAT RENAL MESENCHYMAL TUMORS INDUCED BY NICKEL SUBSULFIDE(1990-09) Higinbotham, Kathleen G.; Hood College Biology; Biomedical and Environmental ScienceRenal mesenchymal tumors were induced in high incidence in male F344 rats by a single intrarenal injection of nickel subsulfide (Ni₃S₂) alone or nickel subsulfide plus iron (Fe°) or magnesium carbonate (MgCarb) (Kasprzak et al., 1990a). The tumors appeared by light microscopy to be comparable in histogenesis to the renal mesenchymal tumors induced by nitrosamines including methyl(methoxymethyl)nitrosamine (DMN-0Me) but were more pleomorphic and had a distinct tendency to metastasize. High-molecular-weight DNA was prepared from the tumors and assayed for transforming activity in NIH 3T3 cells; DNAs from 4 of 16 tumors showed transforming activity. Southern analysis confirmed the presence of the rat K-ras oncogene in the transformed NIH 3T3 clone derived from one of the mesenchymal tumors. Selective oligonucleotide hybridization analysis of polymerase chain reaction (PCR) amplified K-ras gene sequences revealed that 7 of 9 primary tumors induced with N1₃S₂ and Fe° and 1 of 13 primary tumors induced with Ni₃S₂ alone contained exclusively GGT - GTT activating mutations in codon 12. A GGT -. GAT transition was identified in one passaged tumor which had originally contained a GGT - GTT transversion in the primary tumor. The presence of those activating codon 12 point mutations was confirmed by direct sequencing of the PCR amplified K-ras sequences. Sequencing also revealed that there were no other activating mutations at the other codons, 13 and 59-61, that result in activation of the c-K-ras proto-oncogene. These results show that in the rat kidney, Ni₃S₂ carcinogenesis is associated with site-specific activation of a specific cellular proto-oncogene in a fashion consistent with direct interaction of the carcinogen with cellular DNA encoding the target proto-oncogene.Item Measurement of Viral Infection in the Early Stages of Friend Disease(1997-03) Hewes, Suzanne M.; Hood College Biology; Biomedical and Environmental ScienceThe study of the Friend virus complex has provided important insights into cancer and other retroviral diseases. This mixture of retroviruses commences disease almost immediately after inoculation, and the indications may include: splenomegaly, unrestrained proliferation of the reticulum cells, progressive lymphocytosis, hepatomegaly, and erythroblastosis. While Friend disease is well studied, the dynamics of virus replication during the early stages of Friend disease are unknown. Polymerase Chain Reaction (PCR) was used to monitor the kinetics of viral spread by two components of the Friend virus complex. The number of proviruses present in DNA, from the blood and spleen cells of mice infected with the Friend viral complex, was determined. The spread of replication-competent Friend murine leukemia virus (F-MuLV) was found to precede the replication-defective spleen focus-forming virus (SFFVp). Additionally, the detection of Friend viral DNA was first found in the peripheral blood. It was not until later in the course of the disease that the provirus was detected in the spleen. The PCR detection technique allowed for the development of an animal model to monitor the effects of anti-viral compounds in vivo. This system showed that one zinc-finger oxidizing compound, AldrithioI-2, was capable of suppressing viral replication greater than two orders of magnitude, as measured in a six day assay. This study has established a method to detect the presence of Friend provirus, to quantitate the amount of proviral DNA which can project the rise in viral replication, and to directly detect viral replication when zinc-finger oxidizing compounds are administered to an in vivo system.Item THE ROLE OF α₂-MACROGLOBULIN IN THE LIMULUS POLYPHEMUS CLOTTING SYSTEM(1998-05) Herbstritt, Christopher; Hood College Biology; Biomedical and Environmental ScienceThe circulating amebocyte of the North American Horseshoe Crab, Limulus polyphemus, contains a rudimentary immune system that reacts to Gram-negative bacterial endotoxin. Lipopolysaccharide is a purified component of endotoxin and induces an enzymatic cascade in the Limulus blood system, by activating a series of serine protease zymogens, eventually forming a protein clot (Levin, J. and F. Bang 1964; 1968). A variety of commercial endotoxin detection assays have been developed using the Limulus amebocyte lysate, LAL. The pharmaceutical industry uses these products to detect endotoxin in their final products, since endotoxin is a potent fever-inducing pyrogen in humans. β (1-3) glucans, of fungal and yeast origin, also activate the LAL cascade, using an alternate pathway. Current LAL assays detect endotoxin and β (1-3) glucans, but do not allow differentiation between the two. Glucans are not toxic to humans, while endotoxins are toxic and their presence in medical products leads to severe immune responses and septic shock. An assay that could differentiate between the endotoxin and glucan would be useful. In addition to the proteins involved in the LAL cascade, the amebocyte lysate contains a variety of other proteins of unknown function. The third most abundant protein is a serine protease inhibitor named α₂-Macroglobulin, α₂-M. This complex protein is known to bind active serine proteases and remove them from circulation. The role of α₂-M in the LAL cascade is not known. Since α₂-M is a serine protease inhibitor, and the LAL cascade enzymes are serine proteases, it was suspected that removal or inactivation of α₂-M from the LAL system may increase the sensitivity or the rate of the LAL reaction. To address this question, a number of monoclonal antibodies were generated to the Limulus polyphemus α₂-M protein, as a means to study its role in the LAL enzymatic cascade, with minimal alteration of other components of the system. The antibody was characterized by studying cross-reactivity with human α₂-M and a number of mammalian clotting factors. The antibodies were generated by, injecting unreduced Limulus α₂-M into BALB/c mice, collecting the spleens and fusing the spleen cells with mouse myeloma cells. An ELISA screening assay was developed to identify clones producing the antibodies specific for α₂-M. Supernatant material from clones was also tested for specificity using western blot techniques. Clones producing α₂-M antibodies were expanded in mice and ascites was collected. The purification of the antibody was performed using Protein G columns. Large quantities of purified α₂-M antibodies were made from mouse ascites for two of the clones, 7A10 and 2D5. The clones 6E5, 8H12 and 5D6 produced antibodies to α₂-M, but did not produce sufficient quantities of ascites in mice. Antibody isotype determined using ELISA, indicated that all five of the antibodies were the IgG1 isotype. The 7A10 and 2D5 antibodies bound to the α₂-M protein, in its dimer and tetramer forms, on native LAL western blots. ELISA tests with a number of mammalian complement factors showed no cross-reactivity. An ELISA test with human α₂-M showed that the 2D5 antibody did cross-react. When added to the LAL assay, the 7A10 antibody showed almost complete inhibition of the endotoxin mediated pathway, but did not affect the ability of the assay to detect glucan. The 2D5 antibody did not affect either the glucan or endotoxin pathway of the LAL cascade. The antibody 7A10 may be useful in making a glucan specific assay.Item COMMERCIAL VERSUS NON-COMMERCIAL REPELLENTS OF WHITE-TAILED DEER: A FIELD TEST(2012-05) Henson, Deborah; Hood College Biology; Biomedical and Environmental ScienceVegetation damage from white-tailed deer, Odocoileus virginianus, in both agricultural and urban settings, is estimated to be in the millions (U.S. dollars) each year. In response, numerous deer repellents have been developed to deter deer browse on vegetation. This study used 16 captive deer subjects to test six different repellents (i.e., Deer Away Big Game Repellent SprayTm, Hot Pepper WaxTM,Deer GuardTM, Sulfur Repellent, Hot Sauce Repellent, Bitter Repellent) and three modes of action (i.e., fear, pain, taste). The goals of this study were to determine both the effectiveness of individual repellents and the relative effectiveness of commercial repellents versus their non-commercial counterparts. Results showed that Deer Away Big Game Repellent SprayTM, Deer Guard, and non-commercial Sulfur Repellent were the most successful at deterring deer browse, respectively. Given that much of the research on deer deterrents is contradictory and generally disregards non-commercial, repellents I employed a field experiment on captive deer to test multiple repellents, both commercial and noncommercial, over a short period of time. Results from such a controlled design should contribute significantly to the growing body of literature on white-tailed deer control.Item IDENTIFICATION OF AN IMMUNE CORRELATE OF PROTECTION FOR DENGUE(2010-05) Heger, Elizabeth; Hood College Biology; Biomedical and Environmental ScienceA correlate of protection has been defined as a "laboratory result. . that has an accepted positive correlation with the induction by a vaccine of a protective immune response in vaccines" (Markoff 2000). In human clinical trials designed to prove efficacy of a vaccine, one of the goals is to identify the protective immune response that is correlated to clinical protection from disease (Plotkin 2008). Although considerable research has been conducted on the immune response to dengue virus infection, an immune correlate of protection has not been clearly defined. The identification and acceptance of an immune correlate of protection would not only provide a more rapid means of evaluating new dengue vaccines, but would also reduce the size of human clinical studies necessary to prove efficacy of a new vaccine, and very likely decrease the time required to obtain a licensed vaccine. The aims of this proposal include: 1) identification of the LD50 for each of the four dengue strains selected for this research in the AG129 mouse model, 2) establishing whether neutralizing antibody titer is correlated with protection from viremia and mortality following passive transfer of polyclonal antibodies, and 3) using human polyclonal antibodies collected under a minimal risk protocol, compare the protection from viremia and mortality afforded by the human antibodies with that of the mouse polyclonal antibodies to determine whether antibody protection of each are correlated in the mouse model.Item Monoclonal Antibodies to Mouse Mammary Tumor Virus: Antigenic Analysis of Envelope Glycoprotein gp52(1981-01) Heffner, Cathy L.; Hood College Biology; Biomedical and Environmental ScienceTwo sets of hybrid cell lines producing monoclonal antibodies against two different strains of mouse mammary tumor virus (MMTV) were established. The first set (designated MMTV VI and VII) was prepared by the fusion of mouse myeloma cells (NS-1) with the lymphocytes of BALB/c mice which had previously been immunized with the C3H strain of MMTV. Approximately 10% of the hybrid cells from MMTV VI and VII initially plated after cell fusion produced immunoglobulins that reacted in antibody binding assays with C3H MMTV. Forty of these were cloned and six eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from sera and ascites of syngeneic mice inoculated with the hybrid cells. All the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the IgM subclass and three produced IgG₂a. Three serologically distinct specificities were observed when these ascitic fluids were tested against different strains of MMTV. The antigenic reactivities detected were: (1) a type-specific determinant unique to the C3H strain of MMTVs; (2) class-specific determinants shared between C3H and OR MMTV; and (3) a group-specific determinant found on C3H, OR, RIII, and the endogenous C3H (C3Hf). MMTVs. The second set of hybridoma cells (designated MMTV XVII and XXI) was prepared by the fusion of NP-3 mouse myeloma cells with the lymphocytes from the C3Hf mice for XVII and BALB/c mice for XXI. For these fusions approximately 10% of the hybrid cells initially plated showed positive antibody production in binding assays with C3Hf MMTV. Fifty-four of these were cloned and five yielded stable cell lines. Again all monoclones were directed against gp52. One of these cell lines produced IgM, two produced IgG₃, one produced IgG₂b, and one was a direct binder but did not react in immunodiffusion studies. Only two distinct serological specificities were seen with these clones: (1) a type-specific determinant unique to the C3Hf MMTV; and (2) a group-specific determinant shared by C3H, GR, Rill, and C3Hf MMTVs was seen again. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule, and also clearly show that one strain of MMTV can be distinguished from other strains.Item The Effect of Streptococcus pneumoniae Infection on RNA Synthesis by Isolated Rat Liver Nuclei(1980-09) Hauer, Edward C.; Hood College Biology; Biomedical and Environmental ScienceHepatic nuclei were isolated, purified and partially characterized from control and Streptococcus pneumoniae-infected rats. Biochemical and morphological examination showed little contamination by other cell components. Nuclei isolated from the livers of infected rats bound slightly (23%) but not significantly more insulin and showed a marked increase in binding site number when compared to nuclei isolated from control rats. An in vitro system for the incorporation of 1-¹⁴c-uridine-5'-triphosphate (¹⁴c-UTP) into ribonucleic acid (RNA) was developed and characterized. In the presence of hepatic cytosol prepared from either control or infected rats, nuclei isolated from infected rats incorporated significantly more ¹⁴C-UTP into RNA than nuclei isolated from control rats. Similar results were obtained when insulin was substituted for cytosol and the effect was eliminated when cytosol was treated with anit-insulin antibodies. These results suggest that the cytoplasmic factor stimulating incorporation of label into RNA was insulin. These studies suggest that nuclei isolated from infected rats can be stimulated by insulin to synthesize RNA, and that this stimulation may result from an increased number of insulin binding sites on these nuclei.Item ISOLATION OF THE GENE CLUSTER WHICH PRODUCES BORRELIDIN IN STREPTOMYCES ROCHEI(1996-01) Hartland, Cathy L.; Hood College Biology; Biomedical and Environmental ScienceStreptomyces rochei is a Gram positive bacterium which produces borrelidin, an antibiotic active against the causative organism in Lyme disease and which has shown some anti-viral and anti-tumor properties. In our laboratory, production of borrelidin was too low to justify large-scale fermentations. Increased output of borrelidin would make it possible to harvest the antibiotic for further research. One means of increasing borrelidin production is to isolate the responsible gene or gene cluster in a high-copynumber vector. Increased copies of these genes might produce increased amounts of antibiotic. With this aim in mind, the following investigation was carried out. Escherichia coli, Streptomyces lividans, and Streptomyces rochei and their associated plasmid/phage vectors were considered for host/vector systems. S. lividans 66 and pIJ702 were chosen for cloning with size-fractionated gDNA of S. rochei. Two screening organisms, an Alternaria sp. and a Cylindrocarpum sp., sensitive to borrelidin but not to any host metabolites, were chosen and used to identify any borr+ clones. As a positive control for the library construction, the cloned plasmids were isolated and retransformed into a second host cell, demonstrating the transfer of morphological characteristics coded for by the plasmid inserts. The screen for borr+ clones turned up no positive colonies out of 1500 transformants. Attempts to demonstrate a varied genomic library by looking for restriction bands of the cloned plasmids were less clear. A borr- S. rochei mutant was isolated for possible use in future cloning by complementation studies.