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Biomedical subjects

R J Hohman

Publications and source records attributed to R J Hohman.

30 records · Page 2Linked to original sources
PubMed1988

Aggregation of IgE receptors induces degranulation in rat basophilic leukemia cells permeabilized with alpha-toxin from Staphylococcus aureus.

A method has been developed by which rat basophilic leukemia (RBL) cells can be permeabilized to small molecules while maintaining their ability to degranulate in response to aggregation of IgE receptors. alpha-Toxin was isolated from culture supernatants of Staphylococcus aureus by precipitation with (NH4)2SO4 and chromatography on phenyl-Sepharose. The isolated toxin binds to the plasma membrane of RBL cells and polymerizes to form a transmembrane pore that allows small molecules (Mr less than 1000), but not macromolecules, to diffuse freely across the membrane. There was no spontaneous release of the contents of RBL cell secretory granules during permeabilization or subsequent incubations. Substantial IgE receptor-mediated exocytosis occurred in the absence of Ca2+, and degranulation was maximal at 0.1-1.0 microM Ca2+, the physiologically important range of Ca2+ concentrations. Using these permeabilized cells, small molecules (i.e., substrates and inhibitors of various enzymes) normally excluded by the plasma membrane can be introduced into the cell. Moreover, the intracellular concentration of ions (such as Ca2+) can be precisely controlled. This method will allow a detailed examination of the individual biochemical events involved in degranulation of mast cells.

Animals↗
PubMed1986

Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water.

RBL-2H3 cells (a tumor analog of rat mast cells) have plasma-membrane receptors that bind immunoglobulin E, which when aggregated, initiate degranulation. As in other systems, secretion is preceeded by enhanced hydrolysis of inositol phospholipids and by a rise in intracellular Ca2+. Unlike the responses of many other cells, however, both of these earlier events require extracellular Ca2+. The relationship of these events to each other and to the subsequent secretory process is thus unclear. By exposing cells to covalent oligomers of IgE one can demonstrate substantial increases in secretion of histamine by increasing the concentration and size of the oligomers or by using heavy water (D2O) in the medium. We have used such maneuvers to examine the quantitative relationships between aggregation of the receptors and the breakdown of inositol phospholipids, the increase in cytosolic Ca2+ and secretion. Our principal findings were: all treatments that increased secretion, correspondingly increased the changes that precede degranulation. These early events correlated with the degree of aggregation of the receptors even when the stimulatory conditions resulted in maximal secretion. Although the results were insufficient to prove that the hydrolysis of inositol phospholipids is required for the rise in cytosolic Ca2+, the studies with D2O and other observations supported this view. Since a plasma-membrane ion channel for Ca2+ has been implicated in the IgE-mediated rise in cytosolic Ca2+ in RBL 2H3 cells, this in turn suggests a heretofore undescribed role for hydrolysis of inositol phospholipids.

Animals↗
PubMed1985

Inactivation of S-adenosyl-L-homocysteine hydrolase by cAMP results from dissociation of enzyme-bound NAD+.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is inactivated by cAMP and also by 2'-deoxyadenosine, and in both cases, activity is restored by incubating the inactivated enzyme with NAD+. We have previously presented evidence that, despite these similarities, inactivation by these two ligands proceeds by different mechanisms. We have now used a fluorescence technique to quantitate enzyme-bound NAD+ and NADH on S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum, and we have confirmed that cAMP and 2'-deoxyadenosine inactivate by different mechanisms. Whereas inactivation by 2'-deoxyadenosine is due to reduction of the enzyme-bound NAD+ to NADH, incubation of S-adenosyl-L-homocysteine hydrolase with cAMP results in dissociation of the enzyme-bound NAD+. The dissociation is reversible, and reactivation likely occurs by restoration of the initial NAD+ content. This reversible inactivation by cAMP may be a mechanism of controlling biological methylation reactions by adjusting intracellular concentrations of S-adenosyl-L-homocysteine through action of S-adenosyl-L-homocysteine hydrolase.

Adenosylhomocysteinase↗
PubMed1984

S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum is inactivated by cAMP and reactivated by NAD+.

Purified S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum is inactivated when incubated at 25 degrees C with cAMP. Half maximal velocity of the inactivation process occurs at 10 microM cAMP. Catalytic activity is fully restored by further incubation with NAD+, but not with NADP+ or NADH. The enzyme must be preincubated with cAMP or NAD+ to induce inactivation or reactivation, respectively, since neither of these ligands has an effect on the active or inactive enzyme when added directly to the assay. These results suggest a role for cAMP and NAD+ in the regulation of cellular methylation reactions by altering the level of S-adenosyl-L-homocysteine via S-adenosyl-L-homocysteine hydrolase.

Adenosylhomocysteinase↗
PubMed1984

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine.

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.

Adenosylhomocysteinase↗
PubMed1980

Subunit interaction of adenylylated glutamine synthetase.

The glutamine synthetase (GS) activity in Escherichia coli is regulated by a bicyclic interconvertible enzyme cascade which involves the cyclic adenylylation (inactivation) and deadenylylation (activation) of GS on the one hand, and the modulation of these processes by the uridylylation and deuridylylation of Shapiro's regulatory protein on the other. The specific activity of GS in a given metabolic state is determined by the fraction of its subunits that are adenylylated, and this fraction is determined by the concentration of over 40 metabolites. Through allosteric and substrate interactions with one or more of the cascade enzymes, these metabolites alter the rates of the covalent modification and demodification reactions. By means of immunoprecipitation studies with anti-AMP specific antibodies, it has been established that the partially adenylylated glutamine synthetase, which is present in a given steady state, is a mixture of hybrid molecules containing different numbers and possibly distributions of adenylylated subunits. Partial separation of these hybrid mixtures has been achieved by affinity chromatography on Affi-Blue Sepharose columns. From immunochemical studies it is evident that anti-AMP antibodies can react with adenylylated subunits of all molecular species of GS, but that the capacities of these primary antigen-antibody reactions to yield precipitable aggregates is very dependent of the number of adenylylated subunits per molecule, and much less so upon the total concentration of adenylylated subunits present. the studies suggest that precipitability is a function either of the distribution of adenylylated subunits within hybrid species, or of the kinetics of intra- vs intermolecular bivalent interactions.

Glutamate-Ammonia Ligase↗
PubMed1980

Anti-AMP antibody precipitation of multiply adenylylated forms of glutamine synthetase from Escherichia coli: a model relating both concentration and density of antigenic sites with the antibody-antigen interaction.

Sheep antibodies directed against an AMP-bovine serum albumin conjugate exhibit highly specific binding toward AMP. These antibodies bind to the AMP moiety of adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli and to no other antigenic determinant on the protein. E coli glutamine synthetase can exist in variously modified (isomeric) forms that differ with respect to the number (0-12) and the distribution of identically adenylylated subunits [Ciardi, J. E., Cimino, F. & Stadtman, E. R. (1973) biochemistry 12, 4321-4330]. Using this enzyme, together with the AMP-specific antibodies, we have investigated the effects of the total concentration, population density, and topographical distribution of multiple identical antigenic determinants on the antigen-antibody interaction. Stopped flow fluorescence measurements show that the rate and extent of initial binding of the antibodies to the antigen are a function of the total concentration of AMP groups and are independent of the number of AMP groups der dodecamer. However, the rate of lattice formation increases with increasing epitope density, and the maximal amount of glutamine synthetase precipitated is directly proportional to the average number of adenylylated subunits per dodecamer. The data suggest that partially adenylylated enzyme preparations are composed of subpopulations of glutamine synthetase molecules that differ in their tendency to form precipitable aggregates, due presumably to differences in the topographical distribution of antigenic determinants on the surface of the enzyme. The enzyme species that form soluble immune complexes do so possibly due to intramolecular crosslinkage of the bivalent antibodies with adenylylated subunits to the exclusion of intermolecular crosslinkage.

Adenosine Monophosphate↗
PubMed1977

Electron microscopic study of cell surface rings during cell division and morphogenesis of Arthrobacter crystallopoietes.

The whole cell ultrastructure during cell division and morphogenesis of Arthrobacter crystallopoietes was monitored using electron microscopic techniques. Glucose-grown spherical cells were inoculated into succinate-based medium. In this medium, the organism undergoes a morphogenetic cycle consisting of elongation of spheres to rods, exponential growth as rods, and fragmentation of rods to spherical cells. Raised bands or rings that encircled the cells were evident on the cell surface of both sphere- and rod-shaped cells. Many rod-shaped cells possessed two or more rings arranged adjacent to each other in a parallel orientation. At each cell division a new ring was formed on both siblings. However, as predicted by the proposed model of unidirectional cell growth and by maintaining a ring from the previous generation, unequal numbers of rings were observed on sibling cells. Only one ring was visible on most of the spherical inoculum cells, but in some cases a second ring perpendicular to the other ring was observed. Parallel rings were found on spherical cells resulting from fragmentation or reductive cell division of rods during the stationary growth phase. Thus, these spheres could be distinguished from inoculum spheres containing a single ring or perpendicular orientation of rings. The number of rings per cell and arrangement of rings on the cell surface of sibling cells after cell division, but before cell separation, are discussed with respect to cell age, cell division, and sphere-rod-sphere morphogenesis of A. crystallopoietes.

Arthrobacter↗
PubMed1974

A comparison of three methods for eliminating disruptive lunchroom behavior.

THREE METHODS OF CONTROLLING DISRUPTIVE LUNCHROOM BEHAVIORS OF ELEMENTARY SCHOOL CHILDREN WERE COMPARED: basic modification procedures, basic modification procedures plus punishment essays, and basic modification procedures plus mediation essays. During an in-service workshop, six paraprofessional lunch aides received training in these methods to modify three classes of disruptive lunchroom behaviors. They then applied the methods in a counter-balanced design. Fourth- and fifth-grade elementary school pupils were observers and made reliability counts of the target misbehaviors under the various methods. Results indicated that during the periods when aides had been directed to use basic modification procedures plus mediation essays, target misbehaviors were almost totally eliminated and occurred significantly less often than during the periods when they had been directed to use basic modification procedures alone or basic modification procedures plus punishment essays.

Adolescent↗