Hood College Biomedical and Environmental Science
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Item INTERACTIONS BETWEEN GALACTOSE REPRESSOR PROTEIN (GALR) AND THE ALPHA SUBUNIT OF RNA POLYMERASE: REPRESSION AND ACTIVATION OF THE gal PROMOTERS(1995-12) Hanger, Robert R.; Hood College Biology; Biomedical and Environmental ScienceTo study molecular mechanisms of gene regulation at the level of transcription, the gal operon of Escherichia coli was used as a model system. Two overlapping promoters P1 and P2 separated by 5 bp and located on opposite faces of the DNA double helix control transcription of galETKM. DNA templates for in vitro transcription experiments were constructed in order to study protein-protein interactions between the repressor GalR, binding at the external operator OE, and the C-terminal domain portion of the alpha subunit of RNA polymerase (α-CTD) binding at gal promoters under non-looping conditions. Partial repression mediated by GalR was quantitated on mutant templates that had only the Pt promoter intact. Activation mediated by GalR was established on mutant templates that had only the P2 promoter intact. Templates were also constructed to alter the geometry between OE and gal promoters. It was shown that GalR mediated repression occurs at either promoter provided GalR binds at OE located on the same face of the DNA double helix as RNA polymerase binding at the promoter, and it was determined that activation occurs at either promoter provided GalR binds at OE located on the opposite face of the DNA with respect to RNA polymerase. Transcription experiments were then performed using mutant RNA polymerases with tryptophan substitutions at sites from amino acids 261 to 270 of α-CTD). It was shown that the same region on α-CTD known to interact with cAMP receptor protein (CRP) at Class I CRP dependent promoters, and with the UP regulatory element at rrnBP, also interacts with GalR when bound at OE, or interacts with DNA near OE, and that it is this interaction that is responsible for GaIR mediated control of transcription. It is proposed that α-CTD may interact with a 3rd DNA regulatory element overlapping OE, and thus stimulate transcription from galP2. Partial repression at the galP1 promoter may be the result of GalR preventing this interaction, by repositioning α-CTD to a site further downstream, to a location where open complex formation is not favored. Alternatively, activation and repression may be due to conformational changes in RNA polymerase induced by protein-protein interactions between α-CTD and GaIR.Item CALORIE RESTRICTION AND ENDOMETRIOSIS: IS THERE A PROTECTIVE FUNCTION?(2003-09) Handy, April M.; Hood College Biology; Biomedical and Environmental ScienceEndometriosis, defined as the presence and proliferation of endometrial tissue outside the uterus, affects up to 20% of women of reproductive age and can cause severe pelvic pain and infertility. It is well known that endometrial tissue proliferates under the influence of estrogen and that inflammation is most severe when circulating levels are high (late follicular phase of the menstrual cycle). The mechanism(s) leading to the implantation and proliferation of ectopic endometrial tissue in some women, while re-absorption occurs in others, is not known. Current therapy for the disease focuses on controlling the production of estradiol, and in some cases, inhibiting it completely along with reducing the ectopic tissue with periodic abdominal surgery. Understanding the mechanism(s) underlying the initiation and progression of endometriosis may allow for the development of less invasive, targeted therapies. Hypothetically, the incidence and/or progression of proliferative diseases, such as endometriosis, could be reduced if the overall rate of cellular proliferation could be controlled. Calorie restriction (CR), a nutritional intervention that extends lifespan and retards age-related disease in a variety of short-lived species, has been shown to reduce proliferation in lymphocytes as well as liver and mammary tissue. Calorie restriction has also been shown to reduce both spontaneous and induced forms of cancer in rodents. A preliminary report from the Institute on Aging (NIA) longitudinal study of CR in nonhuman primates showed a reduced incidence of naturally occurring endometriosis in CR vs. ad libitum (AL) female rhesus monkeys (22.6% and 11.1%, respectively). Assessment of the entire colony will not be available for many years due to the longer than thirty-year lifespan of rhesus monkeys. Therefore, the purpose of this study was to begin to examine the possible protective function(s) of CR against proliferation of extrauterine endometrial tissue in an established, short-lived model of rat endometriosis. In this study, forty female Fisher 344 rats were divided into two groups: ad-libitum fed (AL) and calorie restricted (CR) that received 40% fewer calories. Half of the animals in each diet group underwent abdominal surgery during which one uterine horn was removed, divided, and autotransplanted onto alternating descending arterial cascades in the mesentery surrounding the small intestine. Sham-operated animals in both diet groups served as controls. The implants were allowed to remain for five to six weeks and were compared for growth during a second abdominal surgery. Body weight was significantly lower in the CR group as was the weight of the remaining intact uterine horn at the time of the second surgery. The percentage of endometrial implants surviving at five to six weeks was not significantly different between the two diet groups. The overall growth of implanted tissue was higher in the CR group. This preliminary investigation produced no clear evidence that proliferation of uterine tissue is slowed by the proposed dietary intervention. Although the results gathered in this study do not confirm our original hypothesis, there are other aspects of endometriosis, primarily the attachment process, which remain to be explored.Item ROLE OF VP40 IN EBOLA ASSEMBLY(2001-06) Hamilton, Elaine; Hood College Biology; Biomedical and Environmental ScienceThe VP40 protein of Ebola virus (EBOV) is thought to be a key component in the assembly of infectious virions. If so, one might predict that when expressed alone, it would form virus-like particles. To test this possibility, we created a plasmid that expressed VP40 under the control of a CMV promoter. Transfection of mammalian cells with this construct showed that VP40 was synthesized as a single protein species that was eventually released into the culture medium. Pulse-chase experiments demonstrated that VP40 protein expressed alone had a rate of release that was comparable to infectious EBOV. Further analysis of the culture medium by density gradient centrifugation revealed that VP40 was released as two distinct forms: one with a density similar to that of authentic virions (1.15 g/cm³) and one with a density consistent with lipid membranes (1.05 g/cm³). Deletion mutants of VP40 identified areas within the protein that are necessary for the protein to mediate its release from cells. EM and immuno-EM studies of VP40-transfected cells showed that VP40 localized at or near the plasma membrane where it induced proliferation, or "ruffling", and formation of empty liposomes. These data suggest that VP40 has the capacity to associate with the host-cell plasma membrane and cause evaginations that are released from the cell as liposomes in the absence of the other viral components. However, with further investigation, we now know that this is not entirely true. VP40 does bind to the surface of the cell and induces proliferation, but the liposomes and "free" protein released from the cells are merely artifact. Electron microscopy (EM) of both isopycnic gradient peaks reveals fib -particles in the light peak and seemingly no particles in the heavy peak. However, additional isopycnic gradients in which the culture media was pre-treated with trypsin eliminated the light peak, and showed the small amount of VP40 protein in the heavy peak was resistant. This suggests that the particles seen in the light peak are artifact of EM or the sucrose gradient. In fact additional experiments show that VP40 is extremely inefficient in budding; rather plays a role in ruffling for viral pre-assembly.Item CHRONIC HIV INFECTION: DEVELOPMENT AND CHARACTERIZATION OF CHRONIC INFECTION MODEL SYSTEMS FOR THE EVALUATION OF ANTIVIRAL AGENTS(1994-05) Halliday, Susan; Hood College Biology; Biomedical and Environmental ScienceMillions of people worldwide are infected with human immunodeficiency virus (HIV). Latent HIV disease in infected patients is characterized as an asymptomatic phase which can last for over 10 years. Recent studies have shown that virus replication in lymphoid tissues from HIV-infected patients continues at a high level throughout the clinically latent phase. Useful model systems of the latent stage of HIV disease in vivo need to be developed to evaluate therapeutic agents which could be of help to the vast number of infected individuals. Several in vitro model systems of latent HIV-infection (i.e., U1, ACH2, OM10.1) have been developed and utilized in the study of chronic/latent HIV infection. Cell lines of this type have been used to investigate the role of inductive and inhibitory signals on HIV replication. However, a good model in vitro system for evaluating agents capable of reducing the infected cell population or inhibiting the spread of virus from cell to cell is currently unavailable. Such cell-based models could be of tremendous value in the study of chronic/latent HIV infection. Little is known about the biological characteristics of chronically infected cell lines. The purpose of this project was to develop model systems of chronically infected cell populations in order to characterize the chronic state of HIV infection in vitro. Once these model systems were established, a group of antiviral agents with diverse mechanisms of action were evaluated. This evaluation included their effect on virus production and their ability to inhibit virus spread by cell-to-cell transmission. Populations of chronically infected MT2, CEM-SS, H9, U937, and CEM-CCRF cells were derived as part of this study. These chronically infected cell populations were biologically and biochemically evaluated in order to define and characterize the infected cells and to compare them with parental uninfected cells. It was determined that the chronically infected cell populations derived from cytopathically infected MT2 and CEM-SS cells include a larger population of nonproductively infected cells than of productively infected cells. Cells derived from noncytopathically infected H9, U937, and CEM-CCRF cells yielded a population of cells which were predominantly productively infected. These chronically infected cells thus served as good models of chronic HIV infection since they continuously produce virus in the absence of any associated cytopathology. A panel of antiviral agents with diverse mechanisms of action was evaluated to determine their effects on chronic HIV infection and the cell-to-cell spread of virus using these cell-based model systems. One compound, SRI 7755, was identified in these studies that was capable of significantly reducing the number of infected cells in a chronically infected population as well of inhibiting the induction of virus expression in latently infected U1 cells. The major goal of this project was to develop and characterized chronically HIV infected cell lines. The identification of compounds effective in inhibiting or suppressing virus production in these chronically infected cell populations was also demonstrated.Item Increased Resistance to Cytomegalovirus in Graft-versus-Host Immunosuppressed F1 Mice by Pre-Immunization(1986-05) Hallam, John A.; Hood College Biology; Biomedical and Environmental ScienceGraft-versus-host (GvH) immunosuppression was induced in (C57BL/10xB10.A)F1 hybrid mice by intravenous inoculation of 30x10⁶ parental (B10.A) spleen cells. This treatment caused the normally resistant F1 mice to become highly susceptible to cytomegalovirus (CMV) challenge delivered 7 or 14 days later (survival below 10%). This susceptibility was reversed by immunization with live CMV by either intraperitoneal (i.p.) or subcutaneous route prior to GvH immunosuppression and challenge (survival greater than 87%). Mice which were immunized by the i.p. route and subsequently immunosuppressed without then being challenged had significantly higher mortality due to recrudescent CMV infection than similarly treated mice that did receive a challenge. These data suggested a boosting effect of the challenge in immunized mice. In vitro cell mediated responses to trinitrophenyl (TNP) modified self and allogeneic histocompatibility antigens were abrogated in all GvH groups 10 days after parental cell inoculation which demonstrated that GvH immunosuppression was reversed only for CMV. The CMV specific humoral antibody (Ab) response was monitored using an enzyme linked immunosorbent assay (ELISA) method. The CMV was highly immunogenic in pre-GvH F1 mice as demonstrated by a rapid rise in IgG Ab titer. In GvH immunosuppressed mice, the titer peaked after parental cell administration and then declined rapidly. The titers were high at the time of challenge and specific Ab may have contributed to defense against challenge. The effect of the post GvH challenge in preventing recrudescent CMV infection could not be explained in terms of protecting Ab since titers declined in both cases.Item AMYLOID PLAQUES IN CHRONIC WASTING DISEASE(1990-09) Guiroy, Don C.; Hood College Biology; Biomedical and Environmental ScienceChronic wasting disease (CWD), a progressive neurological disorder of captive mule deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation of the neuronal perikarya and astrocytic hypertrophy and hyperplasia. Histochemically demonstrable amyloid plaques and amyloid plaques reactive to antibodies prepared against scrapie amyloid in captive mule deer, mule deer hybrids and Rocky Mountain elk naturally affected with CWD are presented. Amyloid plaques in CWD-affected mule deer were congophilic, birefringent and periodic acid-Schiff (PAS) positive. In mule deer hybrids, only occasional PAS-positive plaques were observed. No histochemically demonstrable plaques were observed in CWD-affected Rocky Mountain elk. Antibody raised against scrapie amyloid showed robust immunoreactivity with amyloid plaques in CWD-affected captive mule deer and were found in the cerebral grey and white matter in clusters or in isolation, in deep subcortical nuclei, in all layers of the cerebellum, in areas of extensive vacuolation and in subpial and perivascular regions. In hybrid deer, plaques were rarely observed in cerebellum and subpia. A notable finding in brain sections of CWD-affected hybrid deer and Rocky Mountain elk was a circumscribed collection of scrapie amyloid-immunopositive nuclei with non-congophilic, non-birefringent amyloid deposits located at its center. In elk, plaques were not observed in cerebellum, subpia and subependyma. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Similar scrapie amyloid-immunoreactive plaques are also present in Creutzfeldt-Jakob disease, Gerstmann- Straussler syndrome and kuru in humans. The data presented here corroborate that CWD belongs to the subacute spongiform virus encephalopathies (transmissible cerebral amyloidoses).Item AMMONIA AND NITRATE UPTAKE KINETICS BY THREE SPECIES OF AQUATIC VASCULAR PLANTS(2007-12) Guedes, Marcia; Hood College Biology; Biomedical and Environmental ScienceItem THE DEVELOPMENT OF A WESTERN BLOT (IMMUNOBLOT) ASSAY FOR THE DETECTION OF IgG AND IgM ANTIBODIES TO TREPONEMA PALLIDUM IN HUMAN SERUM(1996-12) Gross, Robert W.; Hood College Biology; Biomedical and Environmental ScienceA Western Immunoblot assay was developed to detect IgG and IgM antibodies to Treponema pallidum, the causative agent of Syphilis, in human serum. A total of 220 human serum samples and 3 cerebral spinal fluid samples were run on the Syphilis Western ImmunoBlot (SWIB) IgG assay and the Fluorescent Treponemal Antibody Absorption (FTA-ABS) assay to determine relative sensitivity, specificity and accuracy. High specificity for the 14, 15 and 48 kDa antigens was demonstrated with FTA-ABS positive samples versus FTA-ABS negative samples. The development of only one of these three bands in the SWIB IgG assay was indicative of a positive sample. Sensitivity, specificity and accuracy were checked further by running 30 human serum samples from a well-defined CDC syphilis panel and 14 potentially cross- reactive autoimmune samples on the SWIB IgG assay. The sensitivity, specificity and accuracy for all 267 samples were 97.4% (111/114), 98% (150/153) and 97.8% (261/267), respectively. A total of 151 human serum samples were tested on the SWIB IgM assay and a commercially available Syphilis IgM Capture ELISA to determine relative sensitivity, specificity and accuracy. A lack of specificity with the 37 kDa antigen was demonstrated. Overall, very few bands were visualized on IgM blots and no definitive positive cut-off was evident. Three samples were equivocal by ELISA and were not included in the calculations. The sensitivity, specificity and accuracy of the SWIB IgM assay were checked further by running 14 potentially cross-reactive autoimmune samples. With the appearance of one band other than the 37 kDa antigen band as indicative of a positive, the sensitivity, specificity and accuracy for 162 samples tested were 46.7% (14/30), 84.8% (112/132) and 77.8% (126/162), respectively. Immunochemical analysis of separated Treponema pallidum antigens immobilized on nitrocellulose strips was conducted using mild periodate oxidation to identify any carbohydrate moieties. The 31, 34 and 41 kDa antigen bands lost reactivity with mild periodate oxidation compared with control strips. These antigens are possible glycoproteins of Treponema pallidum.Item Specificity of the Unlabeled Antibody Hemocyanin Bridge Method to Label Virion And Cell Surface Antigens Using Hyperimmune Serum and Monoclonal Antibodies(1981-05) Gregg, Marybelle; Hood College Biology; Biomedical and Environmental ScienceThe unlabeled antibody hemocyanin technique (UAHT) was evaluated for specificity of detection of retrovirus antigens gp70 and p15(E). UAHT used infected cell monolayers incubated stepwise with primary hyperimmune or monoclonal antibodies, secondary bridging antisera, tertiary anti-hemocyanin sera, and hemocyanin (hey) and was amplified by additional anti-hcy sera and hcy incubations. Further, the detection of p15(E) and gp70 by UAHT was compared with the antibody binding (AB) assay. UAHT using hyperimmune sera detected gp70 viral antigens at dilutions >10³ and was increased five-fold with amplification steps. Surprisingly, the detection of these same antigens decreased to <10³ using monoclonal antibodies in place of the hyperimmune serum. The AB assay however detected viral gp70 and p15(E) using monoclonal reagents: 16-11C1, 19-F8 and 19-IIIA2. The only positive UAHT detection of gp70 was with the monoclonal antibody 16-11C1 and 19-F8 for p15(E). The presentation of the antigenic sites may therefore be different in the AB and UAHT assays. Finally, detection of gp70 and p15(E) was determined by the UAHT assay during virus maturation. Hcy labeling was observed in the stages of virus morphogenesis in retrovirus-infected cell monolayers but not in the NSI/1 myeloma parent cell pellets although these cells contained intracisternal type A and extracellular type C viruses.Item QUANTIFICATION OF PHOTODIMERS FROM ULTRAVIOLET RADIATION-INDUCED DNA DAMAGE IN THE SEA ANEMONE, AIPTASIA PALLIDA USING ENDONUCLEASE SENSITIVE SITES (ESS)(2011-05) Griebel, Jessica M.; Hood College Biology; Biomedical and Environmental ScienceTropical littoral zones offer biologically harmful environments to marine invertebrates due to high levels of exposure to ultraviolet radiation. I documented the extent of DNA damage in the sea anemone Aiptasia pallida under laboratory conditions by applying UVR to live animals as well as pooled DNA that had previously been extracted from A. pallida. Cultured A. pallida from Walsingham Pond, Bermuda were subjected to varying lengths of UVR exposure to quantify DNA damage in the form of number of cyclobutane pyrimidine dimers (CPD's). An endonuclease was applied to those treated with UVR to determine varying amounts of DNA damage. Samples without endonuclease were more significant compared to those treated with endonuclease. Overall, all treatments were not statistically significant at the 0.05 alpha level. Small sample sizes and the inability to extract DNA efficiently without causing damage to the controls, were not found to follow a specific pattern.Item THE ISOLATION AND TRANSMISSION OF Helicobacter hepaticus sp. nov., ISOLATED FROM THE LIVERS AND INTESTINAL CONTENTS OF MICE(1994-07) Gorelick, Peter L.; Hood College Biology; Biomedical and Environmental ScienceIn the fall of 1992 at the National Cancer Institute - Frederick Cancer Research and Development Center (NCI-FCRDC), A/JCr stock mice which showed suppurative skin lesions and treated and untreated A/JCr mice from a long-term toxicologic study were found to have a unique chronic active hepatitis of unknown etiology. No biological agent was known to cause such lesions. A major effort was undertaken to determine the cause of the hepatitis, since all these mice were obtained from the NCI-FCRDC Animal Production Area (NCI-FCRDC-APA). The initial approach was to examine potential environmental toxins. Upon failure to identify any specific environmental cause, a search for a possible biological agent was pursued. In the course of utilizing a Steiner's modification of the Warthin-Starry stain (Steiner's stain), pathologists found what appeared to be a helical organism in the hepatic parenchyma of affected mice. The hepatitis was successfully reproduced in A/J mice obtained from Jackson Laboratories (Jax), Bar Harbor, ME, which had been injected with liver suspensions from affected A/JCr mice. A histopathologic survey employing the Steiner's stain on livers from adult NCI-FCRDC-APA mice demonstrated hepatic lesions in numerous mouse strains (A/JCr, C3H/HeNCr, SJL/NCr, BALB/cAnNCr and SCID/NCr), while hepatic lesions were absent in others (C57BL/6NCr, B6C3F1 and nude [nu/nu]). Hepatic lesions were found more frequently in male mice than female mice. Isolation of the organism was accomplished by culturing the livers of affected A/JCr and SCID/NCr mice and incubating the suspensions on trypticase soy agar plates with 5% sheep blood and brucella blood agar with antibiotics at 37°C under microaerophilic conditions. The organism was motile, catalase positive, oxidase positive and rapidly hydrolyzed urea. The organism was tentatively designated Helicobacter hepaticus sp. nov. Subsequently, Helicobacter hepaticus sp. nov. has been cultured from the intestinal tract and feces of affected mice from the NCI-FCRDC-APA and research holding colonies. To demonstrate infectivity, the organism was injected into unaffected A/J mice from Jax. At several time points livers were aseptically collected, cultured, and a portion submitted for histopathological examination. The hepatitis and presence of the organism was confirmed by histopathology and culture. In a third transmission study, CB.17 SCID mice from Taconic Farms (Tac), Germantown, N.Y., were exposed by direct animal or fecal contact to affected SCID/NCr mice from NCI-FCRDC-APA. Over time the organism and the hepatitis were found to have been transmitted to the CB.17 SCID mice, thus demonstrating that the route of infection was fecal-oral. To date, there are no other organisms known to cause the same type of hepatitis as Helicobacter hepaticus sp. nov.. This novel organism has the potential to provide a much needed animal model for biocarcinogenesis and other human health problems.Item PURIFICATION AND CHARACTERIZATION OF RECOMBINANT TOBACCO ETCH VIRUS PROTEASE(1996-05) Goldstein, Adam Stuart; Hood College Biology; Biomedical and Environmental ScienceThe development of expression systems which permit the overproduction of large amounts of protein is a very important advancement in modern biotechnology. Recent developments in cloning have eased some of the traditional problems of obtaining high yields of pure product. New techniques enable the researcher to express and purify proteins of interest by fusing the expressed protein to a variety of commercially available affinity tags such as, f3-galactosidase, glutathione-S-transferase (GST) (Smith, D. 1988), and polyhistidine peptides (Guan, C. 1987). A new site specific protease, Tobacco Etch Virus protease (rTEV), has been cloned and overexpressed for the use in rapid cloning and purification procedures. In a purification scheme, the protein of interest will contain a TEV cleavage site flanked with six histidines. When the protein is cleaved only a single glycine residue at the amino terminus of the protein remains from the inserted rTEV site. With only one glycine residue left on the purified protein there is less of a chance for incorrect foldings, or for unexpected activities.Item DEVELOPMENT OF A HIV-1 NUCLEOCAPSID (p7) PROTEIN CAPTURE ASSAY(1994-04) Goebel, P. Bradley; Hood College Biology; Biomedical and Environmental ScienceThe p24 antigen capture assay, the current immunoassay used to detect and quantitate human immunodeficiency virus, is an undesirable assay when analyzing plasma or serum from an HIV-1 infected person because anti-p24 antibodies interfere with the assay. Before development of an antigen capture assay that may be useful in analyzing samples from HIV-1 infected people, sera from HIV-1 positive people were tested for antibodies to the nucleocapsid protein of HIV-1, p7. Of 801 HIV-1 antibody positive sera tested, 100 (12.5%) were positive for anti-p7, indicating low anti-p7 seroprevalence in infected persons. An antigen capture assay for p7 was developed and compared to the p24 antigen capture assay in reconstruction experiments. HIV-1 diluted in normal plasma was readily detected by both the p7 and p24 antigen capture assays. The p7 antigen capture assay detected virus diluted in the HIV-1 positive plasma as efficiently as from normal plasma. However, the p24 capture assay was ineffective in detecting virus diluted in HIV-1 positive plasma, even at p24 input concentrations as high as 460 ng/ml. Even though the p7 antigen capture assay currently lacks the sensitivity to detect virus in the plasma if HIV-1 infected people, the reconstruction experiments indicate that with increased sensitivity the assay may prove to be useful in assessing viral quantity in HIV-1 infected individuals. The p7 antigen capture assay will be useful in neutralization studies even at the current level of sensitivity.Item CLONING, SEQUENCING AND EXPRESSION OF THE MEDIUM GENOMIC RNA SEGMENT OF SANDFLY FEVER SICILIAN VIRUS(1995-07) Glass, Pamela J.; Hood College Biology; Biomedical and Environmental ScienceSandfly fever virus Sicilian strain (SFS) is a member of the Phlebovirus genus in the family Bunyaviridae. This virus is the causative agent of an acute, self-limiting flu-like illness lasting 2-5 days. Serologic studies have shown that SFS virus is endemic in southern Europe, the Mediterranean, North Africa, and central Asia which coincides with the distribution of its vector, Phlebotomus papatasi. Epidemics of sandfly fever occur when nonimmune adults, such as tourists or soldiers, enter an area where the virus is endemic. Sandfly fever is of military importance due to its short incubation period, capable of rendering large numbers of nonimmune troops ineffective. The development of a vaccine against SFS virus would allow vaccination of troops prior to entering endemic areas. The phlebovirus genome contains three single-stranded RNA segments designated L (large), M (medium), and S (small) of negative or ambisense polarity. The M segment encodes two viral glycoproteins, G1 and G2, and a nonstructural protein, NSм. Studies of other phleboviruses have demonstrated that the glycoproteins are important for viral infection, pathogenesis, and are the primary targets of the immune response. Analysis of the SFS virus M genomic segment was initiated to determine the sequence and genomic organization for use in the development of both nucleic acid-based diagnostic methods and recombinant vaccines. A series of cDNA clones representing the M genome segment were produced by conventional cloning methods and by polymerase chain reaction amplification. All clones utilized for sequence determination were found to be specific for the SFS virus M RNA segment when used as hybridization probes against SFS virus RNA in northern blot analysis. The M-segment cDNA clones were sequenced by the dideoxy method utilizing both manual and automated procedures. The SFS virus M RNA segment was found to be 4402 nucleotides in length. Computer analysis of this sequence predicted a single open reading frame (ORF) 1341 amino acids in length. Based on comparison of the predicted protein sequence of the SFS M segment with other reported phlebovirus M segments, PCR primers were designed to prepare expression cassettes for producing the viral glycoproteins in recombinant baculoviruses. The downstream glycoprotein region was cloned into Autographa califomica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda (Sf9) cells. The expressed protein was characterized by immunoprecipitation with SFS virus hyperimmune mouse ascitic fluid and was found to be indistinguishable, based on electrophoretic mobilities in denaturing SDS-PAGE, from the authentic SFS virus G2 envelope glycoprotein. These results led to the conclusion that the genomic organization of the SFS M RNA segment is 5'-NSм-G1-G2-3'.Item OVEREXPRESSION OF HUMAN P21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ ARRESTS THE GROWTH OF CHICKEN EMBRYO FIBROBLASTS TRANSFORMED BY INDIVIDUAL ONCOGENES(1998-01) Givol, Iris; Hood College Biology; Biomedical and Environmental ScienceIn normal cells, cell growth and division are controlled by the interplay between proto-oncogenes and tumor suppressor genes. Cancer cells usually have both activated an oncogene and lost a functional tumor suppressor gene. This study addresses the interrelationship between these positive and negative growth regulators. The main focus is on p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹, a cyclin/cdk inhibitor whose expression is regulated by p53. High level expression of the p53 tumor suppressor can block the growth of cancer cells. Wafl/cipl is transactivated by p53 and the p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ protein is itself a suppressor of cell growth. To test the growth suppression effect of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ or p53 on the growth of normal cells and cells transformed by individual oncogenes, we used replication-competent retroviral vectors to induce high level expression of p53 and p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ in chicken embryo fibroblasts (CEF). These are primary cells and by using them we avoid the complications that arise when such experiments are done with established cell lines. This study shows that overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ or p53 arrests the growth of CEF at the G 1 phase of the cell cycle by inhibiting DNA synthesis. We next asked whether the growth inhibition induced by p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ could be overcome by mitogenic signals delivered by a variety of oncogenes. We show that the growth of CEF transformed by v-Src, tf-Ras, c-Mos and c-Myc is inhibited by overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹. This suggests that p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ functions downstream of these oncogenes, suggesting that mitogenic signals converge at the cyclin/cdk complex. To address the interplay between positive and negative signals within the cell cycle machinery, we used this system to test the effects of overexpressing both p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ and E2F1, a subunit of the E2F transcription factor, on the growth of CEF. The E2F transcription factors are released from binding to hypophosphorylated pRb when pRb is phosphorylated by cyclin/cdks. This allows for transcription of S phase genes and progression from G₁ to S phase in the cell cycle. In this system very high levels of E2F1 overexpression cause considerable apoptosis, however the surviving cells still overexpress E2F1. These cells are transformed and their growth is blocked by overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹. These data suggest that p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ might be useful for gene therapy for human cancer. We also show that the apoptosis induced by high levels of E2F1 can be blocked by Bc1-2, an inhibitor of apoptosis.Item A PLATFORM FOR THE SCREENING OF BREAST CANCER-ASSOCIATED POLYMORPHISIMS USING A PRIMER EXTENSION MICROARRAY(2007-04) Gilbert, Joseph T.; Hood College Biology; Biomedical and Environmental ScienceIt is of great societal and scientific importance to be able to identify low penetrance polymorphic loci and the combinations of allelic variation that give the greatest risk to an individual for the development of breast cancer. Understanding about this variation will be beneficial both for diagnosis, prevention and in the development of drug targets for breast cancer. To this end I propose the creation of a diagnostic platform that will help to identify the risk factors associated with known or suspected polymorphic markers that may influence the progression of breast cancer. The platform that I propose would entail the creation of an oligonucleotide microarray consisting of oligonucleotide detection primers that would anneal to genomic DNA from a donor and detect polymorphic mutations among known and suspected breast cancer-associated alleles. Genomic DNA would be taken from a patient and then amplified. The loci of interest would be copied out of the genomic material by using a multiplexing PCR primer set. The amplified polymorphic loci would then be cleaned up and combined to create a hybridization probe for the microarray. Once the probe is hybridized to the microarray, a polymerase will then be added along with fluorescent-labeled dNTPs and the detection primer would be extended using the labeled dNTPs. This extension would only occur when there is a complete nucleotide match between the sample DNA from the patient and the detection primer's 3' end. A microarray scanner should be able to detect the presence of incorporated labeled nucleotides. The data from large scale studies using this platform coupled with the monitoring of an individual's medical history that has been genotyped using this method, could provide both scientists and clinicians with data available to make a better judgment regarding future risk and prevention of this disease.Item MOLECULAR ANALYSIS OF FACTORS AFFECTING LYMPHOMA SUSCEPTIBILITY OF AKXD RECOMBINANT INBRED MOUSE STRAINS(1987-06) Gilbert, Deborah J.; Hood College Biology; Biomedical and Environmental ScienceRecombinant inbred (RI) strains of mice result from the inbreeding of an F₂ generation which itself was produced by crosses between two existing inbred strains. The genomes of individual RI strains represent stable segregant populations resulting from the reassortment of the two progenitor genotypes. The AKXD RI strains have been derived from progenitor strains AKR/J and DBA/2J that differ significantly in lymphoma incidence. As a result of how the genes affecting lymphoma susceptibility have segregated and been fixed in the RI strains, expression of different levels of lymphoma susceptibility, different ages of onset of disease and involvement of different cell types in the tumors have been observed among the AKXD strains. Ten independently derived AKXD RI strains were analyzed for lymphoma susceptibility. Nine strains were found to be highly susceptible to lymphomas although the level of susceptibility varied greatly. Southern blot analysis of the ten different AKXD strains using molecular probes representing the joint regions of the immunoglobulin heavy chain, kappa light chain and T-cell receptor β-chain genes was performed to classify the strains according to predominant lymphoma type. Additionally a histological classification based on the cell morphology scheme of Pattengale and Taylor was used to verify the molecular classifications of stem, Pre-B, B, T and myeloid cell lineages. Of the nine highly lymphomatous strains analyzed, three died primarily of T cell tumors, two died primarily of B-cell tumors and four were susceptible to both B and T cell tumors. Using molecular probes specific for ecotropic proviruses or ecotropic and oncogenic class I mink cell focus forming (MCF) proviruses it was shown that 91% of the 135 tumors analyzed contained detectable somatically acquired proviruses. Generally MCF proviruses were associated with T-cell lymphomas whereas ecotropic proviruses were associated with B-cell lymphomas. The segregation of a dominant gene, Rmcf, which alters the sensitivity of cells to infection by MCF MuLVs, was recently identified by Hartley et al. on mouse chromosome 5. The allele of this gene carried by each strain also correlated with the predominant lymphoma types seen in these tumors. The three strains that died predominantly of MCF virus -associated T cell lymphomas inherited the Rmcfˢ (sensitive) allele from the parental AKR/J strain. AKXD-2 inherited the Rmcfʳ (resistant) allele from the DBA/2J parental strain and lymphomas from this strain contained no detectable MCF proviruses. Using probes homologous to Myc, Pvt-1, Pim-1 and Fis-1, rearrangements were detected in lymphomas and shown to result from viral integration in the respective regions. Proviral integration near Myc, Pvt-1, Fis-1 and Pi -1 were detected primarily in T-cell lymphomas. In three cases rearrangements near Fis-1 and Pim-1 were detected in the same tumor tissue as rearrangements near Myc. Proviral integration near Myb was not detected in any of the lymphomas analyzed. The significance of the genetic loci analyzed will be discussed with respect to their role in the molecular basis of the disease process.Item COMPARING THE COMPREHENSION OF PARENTAL INFORMED CONSENT AND THE CORRESPONDING MINOR ASSENT DOCUMENT(2010-03) Giambarresi, Leah; Hood College Biology; Biomedical and Environmental ScienceThe informed consent process has had a long history which led to the development of explicit requirements regarding the information disclosed to adult subjects in clinical trials. There are very few similar requirements for the information that is disclosed to a minor who participates in the same research. This study aims to examine the difference in the understanding of an adult consent document as compared to its respective child assent document from the same trial. Two document pairs were selected based upon the presence of the 14 required and additional elements of informed consent as described in 21 CFR 50.25. In the first pair ("consistent"), there was very little difference in the number of these elements presented; the second pair ("inconsistent") had a far greater difference. A total of 131 volunteers each read one of the documents and answered a series of written questions designed to analyze their understanding of the trial and their feelings about participation. Results were tabulated, and the difference in understanding between each consent and its respective assent was calculated. Then, the differences in understanding between the two pairs were compared. Analysis showed an approximate 20% difference in understanding between the pairs. Data was also analyzed to determine how the lack of elements in the assent documents would affect the volunteer s willingness to participate. Close to 40% of the individuals in both assent groups who declined to participate in the described trial attributed this decision to the lack of at least one element. This diminished understanding and difference in participation suggest that a reevaluation of the assent process may be useful in order to respect the autonomy of the minor clinical trial participant.Item TRANS-ACTING FACTORS MEDIATING THE DIFFERENTIATION AND HORMONAL CONTROL OF THE SCD1 AND 422(aP2) GENES IN 3T3-L1 CELLS(1994-12) Geiman, Deborah E.; Hood College Biology; Biomedical and Environmental ScienceThe 3T3-L1 cell culture system is a model used to study the regulation of adipocyte differentiation. 3T3-L1 cells grow initially as fibroblastic cells until they reach confluence. After the addition of three agents, isobutylmethylxanthine (MIX), dexamethasone (DEX) and insulin, the cells begin to differentiate into adipocytes. During the differentiation of 3T3-L1 preadipocytes into adipocytes, there is an increase in the expression of many genes characteristic of adipose tissue. The hormonal and differential regulation of two of these genes, 422(aP2) and SCD1, was examined in this work. Both genes contain a consensus C/EBP binding site and both have been shown to be transactivated by C/EBPα. C/EBPα is a transcription factor shown by Northern analysis to follow the expression of other adipocyte-specific genes, such as 422(aP2), during the differentiation of 3T3-L1 cells. The expression of two other members of the C/EBP family of transcription factors, C/EBPβ and C/EBPδ, increase early during the differentiation program. Their expression increases rapidly upon initiation of the differentiation of 3T3-L1 cells and then decreases as C/EBPα expression increases and predominates through the differentiated state. The sequential expression of the C/EBP isoforms may be important in the coordinate expression of adipocyte-specific genes such as 422(aP2) and SCD1. To sort out the mechanism of transcriptional activation of adipocyte-specific genes, the effect of the agents used to initiate the differentiation 3T3-L1 cells was looked at separately. 8-Br-cAMP, which may be used in place of MIX, was found to be the most effective component in promoting differentiation. The addition of cAMP to 3T3-L1 preadipocytes induced an increase in the mRNA and protein level of 422(aP2) between 8-16 hours after addition. Previous work had found the SCD1 mRNA to increase within 6 hours after addition of cAMP, however, this study found the protein level of SCD1 to be unchanged after cAMP treatment. DNase I footprinting analysis using nuclear extracts from preadipocytes treated with 8-Br-cAMP showed enhanced protection at the AP-1 and C/EBP sites of 422(aP2), but did not affect the binding at the SCD1/BP and C/EBP site of the SCD1 gene promoter. In the case of 422(aP2) this would indicate an increase in the binding of the C/EBP family of transcription factors to the C/EBP site, as well as Fos and Jun to the AP-1 site. Electrophoresis mobility shift analysis revealed there was enhanced binding at the C/EBP site of both 422(aP2) and SCD1 after the addition of cAMP. The predominate factors binding were C/EBPβ and C/EBPδ with very little C/EBPα involved. This is not unexpected since C/EBPβ and C/EBPδ are known to respond to agents such as cAMP. To determine the importance of the C/EBP and AP-1 sites in the 422(aP2) and the SCD1/BP and C/EBP sites in the SCD1 gene promoters, base mutations were made in those sites by site-directed mutagenesis. DNase I footprint analysis revealed that the mutations in the C/EBP site of both genes eliminated the binding of nuclear factors to that site. The mutations were also cloned into a CAT expression vector to determine the effect on the regulation of the 422(aP2) and SCD1 genes. When cotransfected into preadipocytes with an expression vector for C/EBPα, all of the SCD1 mutant CAT chimeras, with the exception of one made in the C/EBP site, eliminated transactivation by C/EBPoc. In addition the same mutations lowered the CAT activity of the SCD1 gene when transiently transfected into adipocytes. All the mutations in the C/EBP and AP-1 sites of 422(aP2) eliminated transactivation by C/EBPα in preadipocytes and substantially lowered the expression of 422(aP2) when transfected into 3T3-L1 adipocytes. These results would indicate the C/EBP sites in both genes, the AP-1 site in the 422(aP2) gene and the SCD1/BP site in SCD1 are important for the regulation of these genes during differentiation. However, while the DNA binding sites studied in this work are important, other elements further upstream in the promoters of these genes may also be involved in their regulation during adipocyte differentiation.Item THE TISSUE-SPECIFICITY OF MOUSE Ets-1 REGULATORY REGION IN A TRANSGENIC MODEL(1994-07) Garrett, Lisa J.; Hood College Biology; Biomedical and Environmental ScienceCellular differentiation is the result of the selective expression of gene products that determine cellular phenotype. To identify the cis - acting control elements of developmentally-regulated genes, it is necessary to be able to identify genetic regions that direct tissue- specific expression in a developmental system. Transgenic analysis involves the introduction of cloned genes into mice by pronuclear injection of fertilized eggs. This method allows for a comprehensive analysis of the temporal and tissue-specific expression of a developmentally-regulated gene. Recently, Ets-1, a developmentally-regulated protooncogene, was determined by in situ analysis to be expressed in various tissues during mouse development and at high levels in the lymphoid and vascular structures. The Ets-1 gene product is a DNA-binding protein that may function in the regulation of many genes through interactions with specific nucleotide sequences in gene promoters. To better understand the regulation of Ets-1, further analysis is required to identify tissue-specificity and control regions that regulate expression. In this thesis, 2.4 kb of the 5' flanking region of the Ets-1 promoter was fused to the lacZ gene and microinjected into mouse pronuclei. Transgenic mice expressed the Ets-1/lacZ fusion gene aberrantly in the central nervous system. No detectable lymphoid or vascular expression was observed. However, certain transgenic expression showed positive correlation with the in situ analysis. These results indicate that the "functional" promoter of Ets-1 previously described by several labs (Jorcyk et al., 1991, and Marjeus et al., 1992) is not sufficient to facilitate tissue-specific expression. Therefore, lymphoid and vascular-specific enhancers must still be identified in the molecular organization of Ets-1. Furthermore, negative regulatory elements may be present in Ets-1 that suppress neuronal expression.