Molecular Genetics Commons^™

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 …

Macrophages But Not B Cells From Aged Mice Are Defective In Stimulating Autoreactive T Cells In Vitro, Aruna Seth, Mitzi Nagarkatti, Prakash S. Nagarkatti, Subbarao Bondada, Venkatachalam Udhayakumar

Microbiology, Immunology, and Molecular Genetics Faculty Publications

In the present study the effect of aging on the capacity of Ia⁺ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4⁺ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherant cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC …

Phenotypic And Functional Properties Of B Lymphocytes From Aged Mice, Subbarao Bondada, Joan Morris, Richard J. Kryscio

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Phenotypic and functional properties of B lymphocytes from individual young and old mice of different inbred strains were studied. B lymphocyte subpopulation defined by the ratios of the densities of cell surface IgM and IgD were found to be altered with age. However, such alterations in B cell subsets were found only in 30-40% of the old mice. B cell mitogenic responses to anti-μ and anti-Lyb2 antibodies were decreased in a majority of DBA/2 mice. Proliferative responses to LPS and antiμ were reduced only in a minority of CBA/Ca mice but there was a very good correlation in the responsiveness …

Isolation And Immunological Characterization Of A Group Of New B Lymphomas From Cba Mice, Venkatachalam Udhayakumar, Peter H. Brodeur, M. S. Rajagopalan, Stephen Zimmer, Karen E. Pollok, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

We have isolated and characterized a new series of B lymphomas which occurred spontaneously in a group of CBA N mice that were transferred with spleen or lymph node cells from 24-month-old CBA Ca mice. Tumor cell lines from six CBA N mice that received spleen cells were rescued and designated as BKS-2, BKS-3, BKS-4, BKS-5, BKS-6, and BKS-7. Also, tumor cells from a recipient of lymph node cells were rescued and the resulting cell line was designated BKL. These tumor cells expressed membrane immunoglobulin (μ,κ), major histocompatibility complex Class I and Class II molecules, B220, Lyb8, Fc receptors, J11d, …

A Specific Defect In The Proliferative Capacity Of B Cells From Old Mice Stimulated With Autoreactive T Cells, Prakash S. Nagarkatti, Aruna Seth, Mitzi Nagarkatti, Natarajan Muthusamy, Barbara Rychlik, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B …

Properties Of Anti-Lyb-2-Mediated B-Cell Activation And The Relationship Between Lyb-2 Molecules And Receptors For B-Cell Stimulatory Factor-1 On Murine B Lymphocytes, Subbarao Bondada, Joan Morris, A. R. Baluyut

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to …

Impaired Autoreactive T Cell-Induced T Cell-T Cell Interaction In Aged Mice, Venkatachalam Udhayakumar, Subbarao Bondada, Aruna Seth, Mitzi Nagarkatti, Prakash S. Nagarkatti

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Self-Ia-reactive (autoreactive) L3T4⁺ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4⁺ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-E^d-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4⁺ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to …

Early Events In B-Cell Activation: Anti-Lyb2, But Not Bsf-1, Induces A Phosphatidylinositol Response In Murine B Cells, Stephan A. Grupp, Judith A. K. Harmony, Arthur R. Baluyut, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent …

The Effects Of Strain Differences On The Binding Of Concavalian A To Mouse Sperm, Lisa A. Mckay

Honors Theses

The current study, lectins were used as genetic probes to determine the phenotype of the sperm from different strains of mice. Lectins are proteins of non-immune origin, which bind to specific carbohydrates (McCoy, 1986). Being that virtually all cell membranes contain carbohydrate components, lectins have been used to identify all types of cells. In this study, a biotinylated lectin Concavalian A (Con A) derived from Canavalia ensiformis, was used and binding was detected using an avidin-biotin peroxidase (ABP) complex technique. The object of the current research was to 1) determine if mouse strain differences have an effect on lectin binding, …

Activation Of B Lymphocytes By Monovalent Anti-Lyb-2 Antibodies, Subbarao Bondada, D. E. Mosier

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Small resting B lymphocytes are activated when their cell surface immuno-globulin (Ig) receptors interact with specific antigen or anti-Ig antibodies. Such a receptor-ligand interaction leads to enlargement and proliferation of B lymphocytes. These activated B cells can subsequently mature into Ig-secreting cells in the presence of appropriate accessory cell signals (1-3). However, monovalent anti-Ig antibodies cannot induce B cell activation even though they can bind to the surface Ig (4-7). It has thus been concluded that membrane Ig has to be cross-linked to induce early steps in B cell activation (membrane depolarization; 8) as well as later steps (proliferation). T …

Role Of A Nonimmunoglobulin Cell Surface Determinant In The Activation Of B Lymphocytes By Thymus-Independent Antigens*, Subbarao Bondada, Donald E. Mosier, Aftab Ahmed, James J. Mond, Irwin Scher, William E. Paul

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized …

Tritium (3h) Radiolabeling Of Protein A And Antibody To High Specific Activity: Application To Cell Surface Antigen Radioimmunoassays, Ronald L. Wilder, Catherine C. Yuen, Subbarao Bondada, Virgil L. Woods, Cornelius B. Alexander, Rose G. Mage

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Staphylococcal protein A and several different immunoglobulins have been radio-labeled to high specific activities (> 106 cpm/pg) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.

Inactivation Of Brain Hexokinase By An Adenosine 5'-Triphosphate Analog, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

An analog of ATP, 6-mercapto-9+nribofuranosylpurine 5’-triphosphate (SH-TP), functions as a phosphoryl donor for the reaction catalyzed by hexokinase. Incubation of the enzyme with millimolar concentrations of this reagent led to its rapid inactivation. However, the corresponding monophosphate derivative of this reagent was much less effective. A plot of the initial rate of inactivation versus the concentration of SH-TP exhibited saturation, suggesting the formation of a reversible complex between the enzyme and SH-TP prior to the inactivation reaction. The dissociation constant of this enzyme-inhibitor complex was the same as the K, of this reagent with respect to hexokinase in the phosphoryl …

Reaction Of Brain Hexokinase With Tetranitromethane: Oxidation Of Essential Thiol Groups, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Treatment of bovine brain mitochondrial hexokinase with a fivefold molar excess of tetranitromethane (TNM) at pH 8.0 results in a complete loss of activity of the enzyme. Spectral measurements and amino acid analysis showed that only cysteinyl residues were modified by TNM under these conditions. The products of the reaction of TNM with the enzyme were cysteic acid and disulfides as found by amino acid analysis of the carboxymethylated hexokinase. Failure of 2-mercaptoethanol to reverse the effect of TNM suggested that the thiol group that is oxidized to cysteic acid is the one essential for enzyme activity. Substrates glucose and …

Molecular Weight Determinations Of Some Salmonella Potsdam Bacteriophage Dna's, Winston H. Richards

Loma Linda University Electronic Theses, Dissertations & Projects

Two group A phages, P9c/2 and P4/2, were studied. Their density, sedimentation velocity, and biochemical properties were compared. Their densities were determined by density gradient centrifugation in 38.3% cesium chloride. Sedimentation velocity was used to determine their molecular weights. To accomplish this, the DNA of P9c/2 and P4/2 was labeled with tritium and T₂ DNA, labeled with ³²P, was used as a standard. The DNA of P9c/2 and P4/2 was separately mixed with T₂ DNA and centrifuged in a 0-15% sucrose gradient. The molecular weights were calculated using the formula D₂/D₁= (M_{2 …}

Biochemical Properties Of Salmonella Potsdam Bacteriophage P4/2, Clyde L. Roggenkamp

Loma Linda University Electronic Theses, Dissertations & Projects

Over 90% recovery of phage P4/2 was realized with differential centrifugation concentration and purification, whereas only about 16% was recovered from the aqueous two-phase polymer - Genetron - ECTEOLA-cellulose column system. Purity of preparations was greatest with the density gradient centrifugation (A₂₆₀ = 2.66 and A₂₈₀ = 1.80 per 10¹² plaque-forming units). With the chemical system of concentration and purification followed by an ECTEOLA anion exchange run, the A₂₆₀ was 6.6 and the A₂₈₀ was 4.3.

Three tests were employed to determine the configuration of P4/2 DNA: (1) a formalin denaturation test, (2) a thermal …