Microbiology Commons^™

The Ability Of Simian Virus 40 Large T Antigen To Immortalize Primary Mouse Embryo Fibroblasts Cosegregates With Its Ability To Bind To P53., Jiyue Y. Zhu, Marina Abate, Philip W. Rice, Charles N. Cole

Dartmouth Scholarship

The large T antigen encoded by simian virus 40 (SV40) plays essential roles in the infection of permissive cells, leading to production of progeny virions, and in the infection of nonpermissive cells, leading to malignant transformation. Primary mouse embryo fibroblasts (MEFs) are nonpermissive for SV40, and infection by wild-type SV40 leads to immortalization and transformation of a small percentage of infected cells. We examined the ability of an extensive set of mutants whose lesions affect SV40 large T antigen to immortalize MEFs. We found that immortalization activity was retained by all mutants whose lesions are located upstream of codon 346. …

Mapping The Transcriptional Transactivation Function Of Simian Virus 40 Large T Antigen., Jiyue Y. Zhu, Philip W. Rice, Michele Chamberlain, Charles N. Cole

Dartmouth Scholarship

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T …

Strain-Specific Neutralizing Determinant In The Transmembrane Protein Of Simian Immunodeficiency Virus, Toshiaki Kodama, Dawn P. Wooley, Daniel P. Silva, Fulvia Dimarzo Veronese, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a …

Selection Of Genetic Variants Of Simian Immunodeficiency Virus In Persistently Infected Rhesus Monkeys, Dawn P. Wooley, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in …

The Growth Of Simian Virus 40 (Sv40) Host Range/Adenovirus Helper Function Mutants In An African Green Monkey Cell Line That Constitutively Expresses The Sv40 Agnoprotein., Terryl P. Stacy, Michele Chamberlain, Susan Carswell, Charles N. Cole

Dartmouth Scholarship

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of …

Maternal Immunomodulation Of Neonatal Alloantigen Response, Leh Chang

Loma Linda University Electronic Theses, Dissertations & Projects

Remarkable success has been achieved in the transplantation of allogeneic cardiac grafts into newborn infants at Loma Linda University Medical Center. The superior graft survival rate documented in these patients has not correlated with the degree of immunosuppression rendered, or the selection of genetically matched donors. However, the clinical success has correlated with the age of the recipient at the time of receiving a transplant. Patients receiving an allograft within the first few weeks of life are unique in that they seem to accept the alloantigens of their cardiac graft while responding aggressively to antigens in their environment. These observations …

Characterization Of Hsd::Mudx128 Operon Fusion Mutant Of Escherichia Coli K-12, Marjorie Ann T. Reyno

Loma Linda University Electronic Theses, Dissertations & Projects

The study of the regulation of gene expression in complex genes can be facilitated by the use of operon fusions which place a well characterized lacZ gene under the control of a promoter of interest. An hsd::MudX128 operon fusion mutant of E. coli K-12 isolated by Prakash (1986) was observed to have a high β-galactosidase activity (1000 units), ten to twenty times higher than other hsd::MudX mutants. This β-galactosidase activity was suppressed to a very low level (25 units) by the introduction of an F’ plasmid, F'101, which carries the 98 min to 2 min region of the …

Mechanism Of Escape Of Endogenous Murine Leukemia Virus Emv-14 From Recognition By Anti-Akr/Gross Virus Cytolytic T Lymphocytes., Hillary D. White, Michael D. Robbins, William R. Green

Dartmouth Scholarship

It was previously shown that spleen cells from endogenous ecotropic murine leukemia virus emv-14+ AKXL-5 mice fail to stimulate an anti-AKR/Gross virus cytolytic T-lymphocyte (CTL) response in a mixed lymphocyte culture with primed C57BL/6 responder spleen cells, whereas spleen cells from AKXL strains carrying the very similar emv-11 provirus do stimulate a response (Green and Graziano, Immunogenetics 23:106-110, 1986). We wished to determine whether the lack of response with AKXL-5 spleen cells was at the level of recognition between effector cell and target cell and whether the relevant mutation was within the emv-14 provirus. It is shown here that EMV-negative …

Simian Virus 40 Host Range/Helper Function Mutations Cause Multiple Defects In Viral Late Gene Expression., Terryl Stacy, Michele Chamberlain, Charles N. Cole

Dartmouth Scholarship

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three …

Significance Of Premature Stop Codons In Env Of Simian Immunodeficiency Virus, Toshiaki Kodama, Dawn P. Wooley, Yathirajulu M. Naidu, Harry W. Kestler Iii, Muthiah D. Daniel, Yen Li, Ronald C. Desrosiers

Neuroscience, Cell Biology & Physiology Faculty Publications

The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIV_mac). The SIV_mac251 and SIV_mac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIV_mac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). …

Linker Insertion Mutants Of Simian Virus 40 Large T Antigen That Show Trans-Dominant Interference With Wild-Type Large T Antigen Map To Multiple Sites Within The T-Antigen Gene., Jiyue Y. Zhu, Charles N. Cole

Dartmouth Scholarship

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function …

In Vitro Evaluation Of Cancer Patient Immune Responses Following Infusion Of Radiolabeled Murine Monoclonal Antibody, Kelly A. King

Loma Linda University Electronic Theses, Dissertations & Projects

Several monoclonal antibody (MAb) types derived from mice have been developed specifically for the use of targeting human tumors. The specificity of these murine MAb’s for their respective tumor antigens is very high making them potentially good immunotherapeutic agents for cancer treatment and diagnosis. A limiting factor in using these murine MAb’s in patients is the development of human anti-mouse antibody (HAMA). The presence of HAMA may reduce the effectiveness of MAb for tumor targeting. If patient sensitization could be quickly detected following the initial infusion of MAb, then future infusion could be altered or terminated to prevent further sensitization. …

Determination Of Antibody Levels In Periapical Lesions Against Sixteen Oral Microorganisms By The Elisa Technique, Sammee Lee Jones

Loma Linda University Electronic Theses, Dissertations & Projects

The root canal system, which includes the periapical region of the tooth, may become infected by several types of microorganisms which are normally associated with these tissues. A bacteria-specific antibody response may occur at the local level, where various classes of immunoglobulins have been found and reported in the literature. The purpose of this study was to quantitatively measure locally produced immunoglobulin' levels in periapical lesion abscess materials and to determine the specific reactivity of these antibodies to several microorganisms which are normal flora, as well as being previously associated with the development and pathogenesis of endodontic infections. Periapical lesions …

Absence Of A Structural Basis For Intracellular Recognition And Differential Localization Of Nuclear And Plasma Membrane-Associated Forms Of Simian Virus 40 Large Tumor Antigen., Donald L. Jarvis, Charles N. Cole, Janet S. Butel

Dartmouth Scholarship

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- …

Two Separable Functional Domains Of Simian Virus 40 Large T Antigen: Carboxyl-Terminal Region Of Simian Virus 40 Large T Antigen Is Required For Efficient Capsid Protein Synthesis., Joanne Tornow, Maryellen Polvino-Bodnar, George Santangelo, Charles N. Cole

Dartmouth Scholarship

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T …

Cell Mediated Immunity By Cytotoxicity Assay And The Effect Of Corynebacterium Parvum And Radiation On Mice Bearing Herpes-Induced Tumors, Robert B. Stagg

Loma Linda University Electronic Theses, Dissertations & Projects

Cell mediated immune (CMI) cytotoxic reactivity of Balb/c mice against H238 cells, a Herpes simplex virus (HSV) Type 2 - induced sarcoma, was measured by the ¹²⁵IUdR release assay. The Balb/c mouse response to the growing HSV-induced tumor, treated with radiation and , was measured by survival rate, tumor growth and immune cytotoxicity of spleen and peritoneal exudate cells (PEC) as determined by ¹²⁵IUdR release assay. Subcutaneous (s.c.) inoculation of 1 x 10⁶ H238 cells (high dose) produced progressive tumor growth while s.c. inoculation of 1 x 10⁴ H238 cells (low dose) produced no tumors. A …

Some Aspects Of Carbohydrate Metabolism In The Cercariae Of Schistosoma Mansoni Sambon, 1907, A. Eugene Dunham Jr.

Loma Linda University Electronic Theses, Dissertations & Projects

Cercariac of Schistosoma mansoni were studied for glycogen utilization, uptake of glucose from solution, lactic acid production, and respiration.

Using the anthrone method of determination of carbohydrate, the amount of glycogen used by a single cercaria in 4 hours is determined as 43.2 x 10^-4 μg ± 6.6 x 10^-4 μg.

Glucose was not observed to have been used from solution under the conditions of the experiment, even in the presence of trehalose. The determinations were made by the Glucostat method.

After deproteinization and desugarization, aliquots of supernatant from cercarial suspensions were analyzed for lactic acid using p-phenylphenol …

The Effect Of Varying Protein Intake On Trichinella Spiralis Infection In Mice, Clare K. Kwan

Loma Linda University Electronic Theses, Dissertations & Projects

Three hundred Swiss Webster albino mice three weeks of age were placed on low (8%), medium (24%), high (48%) protein levels and a commercial laboratory animal feed, Purina Chow for various lengths of time: (1) simultaneously with infection, and four weeks thereafter, (2) three weeks before infection and throughout the four-week infection period, and (3) six weeks prior to infection and during the infection period. The protein source was casein. Sixty of the 300 mice used served as noninfected controls, and the other 240 were each infected with 400 washed, suspended Trichinella spiralis larvae obtained from a rat by the …

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses, Catherine M. Crosby, William E. Matchett, Stephanie S. Anguiano-Zarate, Christopher A. Parks, Eric A. Weaver, Larry R. Pease, Richard J. Webby, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors …