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).     

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)     

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.     

Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.     

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Characterization of the biomass of the stems of sweet sorghum

In this study the amount of glucose, sucrose and fructose was determined in the water soluble fraction while cellulose, hemicellulose, pectin and lignin contents were determined in the alcohol insoluble fraction after hydrolysis. Stalks of sweet sorghum (Sorghum vulgare L., var. saccharatum) cv. Vespa, Soave, Roce and MN 1500 at the physiological ripeness stage were used. The results of the analysis of variance with the least significant difference method (LSD, = 0.05) show that cv. Vespa and Roce have a significantly higher total amount of glucose, fructose and sucrose and at the same time, a lower cellulose, pectin, hemicellulose and lignin content then cv. Soave and cv. MN 1500.     

Ascorbic acid as a factor controlling "in vivo" its biosynthetic pathway

The capacity of ascorbic acid biosynthesis in potato tuber tissue is closely correlated with the ascorbic acid content of the cells: the lower the endogenous content of ascorbic acid, the greater its biosynthesis. At the highest level of ascorbic acid found in the cells, the biosynthetic capacity is virtually zero. In these conditions, adding glucose (the first precursor of ascorbic acid) has no effect whatsoever, whereas adding galactono-gamma-lactone (the last precursor) induces a high rate of ascorbic acid synthesis. It is suggested that AA biosynthesis is subject to a regulatory mechanism "in vivo" which controls an initial step in the biosynthetic pathway. The last step in this pathway, catalyzed by galactone oxidase, is never blocked and, moreover, its activity is greater than that of the preceding steps.     

Reaction of Woodward''s reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.