Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.     

Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the kinetics of ovine follitropin agonist-antagonist interactions with pig ovarian membranes

The binding of 125I-labeled ovine follitropin (oFSH) and 125I-labeled deglycosylated ovine follitropin (DG-oFSH) to porcine granulosa cell membranes was studied at equilibrium and nonequilibrium binding conditions and statistically analyzed. Saturation and competition binding experiments revealed homogeneity in the population of binding sites labeled with 125I-oFSH, having a pK estimation of approximately equal to 10. 125I-DG-oFSH similarly interacts with a single uniform class of receptors of equal affinity (pK approximately equal to 10) and binding capacity as oFSH. In contrast, displacement experiments using 125I-DG-oFSH as tracer and unlabeled oFSH as competing ligand demonstrate slope factors less than unity, suggesting apparent heterogeneity of sites not observed with 125I-DG-oFSH vs. DG-oFSH competition experiments. Under these conditions, it appears that FSH binds to two sites in near equal proportion but of unequal affinities. The total specific binding capacities of these sites equal those observed in 125I-DG-oFSH/unlabeled DG-oFSH competition experiments. Analysis of oFSH association kinetics at 37 degrees C by curve-fitting methods is best explained by a biexponential rate equation describing a fast and a slow association component that are equally distributed. DG-oFSH demonstrates a disproportionately greater amount of fast vs. slow binding component. The binding half-times for each component of oFSH and DG-oFSH are similar, i.e., minutes for the fast and hours for the slow t 1/2 times. At 37, 25, and 4 degrees C, DG-oFSH exhibits greater velocity of binding to the receptor than oFSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.     

Sarcomere length uniformity determined from three-dimensional reconstructions of resting isolated heart cell striation patterns.

A- and I-band striation positions have been obtained, three-dimensionally reconstructed, and statistically analyzed from the volumes of resting isolated heart cells. Striation patterns from optically discrete subvolumes are imaged along the length of these myocytes with a computer-interfaced optical microscope imaging system. Planar striation maps are reconstructed by the computer from sequentially obtained striation pattern images displaced across the width or depth of the cell in controlled steps. Multiple planar maps are combined to form full three-dimensional (3-D) reconstructions that illustrate the sarcomeric structure and ordering throughout the volume of the cell. These reconstructions demonstrate a high degree of striation registration throughout most regions of cardiac cells. The striation registration is often slightly (less than 10 degrees) skewed across the width or depth of nearly every cell and is occasionally disrupted between adjacent groups of sarcomeres. These disruptions in registration are always associated with the locations of the nuclei. Rigorous statistical analyses indicate small volumetric regions of the cell delineated by these disruptions can have significantly (0.014-0.113 micron) shorter or longer average sarcomere length periodicities. Unlike skeletal muscle "fibrillenstruktur," these data from cardiac cells exhibit no evidence of helical packing schemes for sarcomere order. These observations suggest that the relatively large nuclei displace and disrupt the normal registration of the sarcomeres, which is probably mediated by internal cytoskeletal structures.     

Effect of a broad-host range plasmid on growth dynamics of Escherichia coli and Pseudomonas putida

Host-plasmid interactions were studied for the broad-host range plasmid, pTJS26, a derivative of RK2. To isolate host and plasmid contributions to the growth dynamics and plasmid stability, separate experiments were performed with host and recombinant cells for two different gram-negative hosts, Pseudomonas putida and Escherichia coli, at two different temperatures, 30 and 37 degrees C. At the lower temperature (30 degrees C) the growth kinetics were not affected by the plasmid, but plasmid instability was observed. At the higher temperature (37 degrees C) growth rates and yields were lower than that for the hosts, but the plasmid was stable. This behavior can be explained by a combination of two phenomena. First, the copy number control mechanism may be temperature sensitive and, second, plasmid segregation may be inefficient. For both E. coli and P. putida the growth dynamics of the recombinant system was dictated by the presence of the plasmid.     

Observation on a 65-kilodalton protein isolated from kanagawa positive strains of Vibrio parahaemolyticus

Crude hemolysin from four KP+ strains of Vibrio parahaemolyticus belonging to serotype 02:K3 exhibited a major protein band (molecular weight, 65 kilodaltons (kDa] in addition to a previously known thermostable direct hemolysin band (molecular weight, 21 kDa) in SDS - polyacrylamide slab gel electrophoresis. These strains showed maximum virulence leading to 100% mouse lethality within 2-6 h. It is hypothesized that this 65-kDa protein may play a vital role in the pathogenesis of the disease caused by V. parahaemolyticus.     

Flow cytometric analysis of T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia: correlation with clinical stage

Several authors have studied the T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia (B-CLL), but previous studies were performed after preceding enrichment procedures, which are known to cause selective losses of certain subpopulations. To correct for this deficiency we used flow cytometric analysis, which enabled us to measure subpopulations directly on total blood samples. We studied T-lymphocyte subsets with OKT monoclonal antibodies in 45 patients with B-CLL. Serum levels of IgG, IgA and IgM were assayed simultaneously and findings were correlated with clinical stage (Rai classification). The absolute number of CD4-positive cells decreased in more advanced Rai stages, while the absolute number of CD8-positive cells increased, resulting in a progressive reduction in CD4/8 ratio. Results from patients in stages with equal prognosis (Rai I and II, Rai III and IV) were similar and when these results were grouped the observed differences were highly significant and clearly correlated with all prognostic groups.