The lymphocyte restimulation system: Evaluation by intra-HLA-D group priming

Homozygous typing cells (HTC) were primed, using responding and stimulating lymphocytes of the same HLA-D groups. These intra-HLA-D group primings showed strong specific responses. Restimulation by HLA-D heterozygous and homozygous cell panels showed no correlation between the restimulating determinant and HLA-D. On the other hand, an unrelated individual, not carrying Dw4 and primed to Dw4 HTC, is restimulated by three of four Dw4-HTC. Thus, one non-HLA-D-associated restimulating determinant and another HLA-D-associated determinant could be identified. The differences among the four Dw4 HTC recognized in secondary MLC could reflect either recognition of separate gene products or recognition of separate determinants on the same gene product.     

Nitrogen Oxyanion-dependent Dissociation of a Two-component Complex That Regulates Bacterial Nitrate Assimilation

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.     

Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias.

The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of skeletal dysplasias (dwarfing disorders) characterized by abnormal epiphyses, with and without varying degrees of metaphyseal irregularities, flattened vertebral bodies, and myopia. To better define the underlying cause of these disorders, we have analyzed the collagens from costal cartilage from several of these patients, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) of intact chains and cyanogen bromide (CNBr) peptides and amino acid analysis. In almost all of the patients in this study group, the type II collagen exhibited a slower electrophoretic mobility when compared with that in normal controls. The mobility of many, but not all, of the CNBr peptides was also retarded. Peptides near the amino terminus were almost always altered, while the mobility of peptides close to the carboxyl terminus were normal in all but the severely affected cases. Analysis of the CNBr peptides on an HPLC sieving column confirmed that the electrophoretically abnormal peptides were of a higher molecular weight than were control peptides. Amino acid analysis indicated that the abnormal collagens have a higher ratio of hydroxylysine to lysine than does control collagen, suggesting that overmodification may be involved in the altered mobility. Our results are consistent with a defect in the collagen helix that results in overmodification of the molecule from that point toward the amino terminus. We propose that some forms of SED and SEMD are associated with abnormalities in type II collagen that results in delayed helix formation and consequent overmodification of the collagen. Cases of SED fit onto a continuous spectrum of clinical severity that correlates positively with both the extent of alteration and the proximity of the defect to the carboxyl terminus.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Long term regulation of nucleoside transport by thyroid hormone (T3) in cultured chromaffin cells

The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-l-thyronine (T₃) throughout the prolonged period studied. The V_max values of this transport obtained in absence and presence of 1 M T₃ were 36.21±2.1 and 44.17±3.5 (means±SD) pmol/10⁶cells/min respectively for 26 hours incubation-time with the hormone. The K_m values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [³H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 M T₃ for 26 hours incubation-time. The values of binding sites per cell were 33,500±3,000 and 40,153±3,700 in absence and presence of T₃ respectively, without changing the K_d constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9±2.8 and 24.3±2.1 hours both in the presence or absence of T₃ respectively. When cells were incubated in the presence of both T₃ and cycloheximide, not only the activatory effect of T₃ was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T₃ activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism.     

Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene.

Virus specific, major histocompatibility complex-restricted, cytotoxic T lymphocytes (CTL) generated in Fischer strain rats infected with human adenovirus type 5 (Ad5) were found to recognize antigenic determinants encoded within the Ad5 early region 1A (E1A) gene. Preliminary mapping studies suggest that the E1A CTL epitopes are encoded within the regions between bp 625 to 810 and 916 to 974 in the first exon of this gene. These epitope-coding regions occur within subregions of E1A that are conserved functionally, and to some extent structurally (approximately 50% sequence homology), among adenoviruses of different groups. Nevertheless, Ad5-specific CTL lysed only targets infected with adenoviruses of the same group (group C; e.g., Ad2) and not targets infected with adenoviruses of different groups (groups A, B, and E). These results suggest that virus-specific CTL may limit adenoviral dissemination by destroying virus-infected cells at an early stage in the viral replicative cycle, during E1A gene expression. Expression of other adenovirus genes does not appear to be required to target infected cells for elimination by CTL.     

Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.