Role of serum in the intracellular killing of staphylococci in rabbit monocytes

Shayegani, Mehdi G. (U.S. Veterans Administration Hospital, Philadelphia, Pa.), and Stuart Mudd. Role of serum in the intracellular killing of staphylococci in rabbit monocytes. J. Bacteriol. 91:1393-1398. 1966.-Although some intracellular killing occurs in rabbit monocytes with heated normal serum or even in monocytes washed three times with Hanks' solution and with staphylococci not exposed to serum, efficient killing of coagulase-positive Staphylococcus aureus cells in the mononuclear phagocytes of rabbits is shown to require heat-labile components of serum. The effect of serum in promoting phagocytosis and intracellular killing may be exhibited either by presensitization of the staphylococcal cells before contact with leukocytes or by the presence of serum in the phagocytic system. Under any conditions studied the rate of intracellular killing of S. aureus is very slow.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

A computer-controlled all-tantalum stopped-flow microcalorimeter with microjoule resolution

A new all-tantalum differential stopped-flow heat-conduction microcalorimeter with microjoule resolution has been developed. The instrument consists of two matched channels, each of which has two reagent inlet lines. A computer is used to process the data and control the syringe drive system which runs the samples through the calorimeter. The reagents are mixed in 0.6 s in ratios of 1:1, 1:2, 1:2.5, 1:4, 1:5, or 1:10. The priming volume from the loading port to the mixer is 1 ml and the reaction volume of the detection tube is 160 microliters. The instrument has a sensitivity of 1.60 J/V.s and a differential baseline stability of 100 nJ/s (p-p) over a 4 h period. The sample size can be reduced to 27 microliters with only a 12% loss in sensitivity. With an electrical step power input, the 10-90% response is 40 s. By using a data decomposition scheme, the response time can be improved to 1 s which allows the direct measurement of moderately fast reaction kinetics. With water/water mixes, differential heats of mixing are typically (+/-) 2 microJ with a standard deviation of (+/-) 2.5 microJ. Reaction heats in the 20-50 microJ range can be measured with a standard deviation of (+/-) 3 microJ. A fast reaction, e.g. HCl dilution, can be completed in 150 s. When loading and priming times are included, 25 reactions can be completed in 120 min. A chilled water jacket is used to allow operation over a temperature range of 4 degrees C to 50 degrees C.     

Isolation and characterization of the beta and epsilon subunit genes of mouse muscle acetylcholine receptor

The genes coding for the beta and epsilon subunits of the mouse muscle nicotinic acetylcholine receptor (nAChR) were mapped by Southern blot analysis, and the entire loci for both genes cloned. The results indicate that they are single-copy genes. Both were sequenced to determine their size and structural organization. The beta subunit gene spans approximately 8 kilobases and is organized into 11 exons. A region containing cysteines, which are thought to form a disulfide bond and which are highly conserved, is encoded by one exon in all muscle acetylcholine receptor genes with the exception of the beta subunit gene, where it is split into two exons. The epsilon subunit gene spans 4.3 kilobases and contains 12 exons; it has the same structure as the gamma and delta nAChR genes. The intron-exon boundaries and exonic organization of the five known nAChR genes were compared. The analysis showed that the first 4 exons and the last exon of all muscle and brain nAChR subunit genes have the same boundaries, with the exception of a nAChR-related gene in Drosophila.     

Threonine Synthase of Lemna paucicostata Hegelm. 6746

Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine γ-synthase, establishing the separate identity of these enzymes. Compared to cystathionine γ-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O-phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The K_m for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet.     

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase)

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.