Inhibition of soil urease activity by aminocresols

Summary The effect on soil urease activity of five aminocresols, at concentrations of 5–100 g/g soil, was examined in the laboratory. Two compounds, 4-amino-o-cresol and 4-amino-m-cresol, significantly inhibited urease activity. The efficacy of 4-amino-o-cresol was compared with that of phenylphosphorodiamidate (PPDA), a known inhibitor, in three U.K. soils. At 50g/g soil 4-amino-o-cresol was as inhibitory as an equivalent concentration of PPDA in a soil with low urease activity, but was less inhibitory in two soils with high urease activity.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

sar1, a gene from Schizosaccharomyces pombe encoding a protein that regulates ras1.

Proper ras1 function is required for normal sexual function in the yeast Schizosaccharomyces pombe. We have found a gene in S. pombe, sar1, that encodes a product capable of regulating ras1 function. sar1 is a member of an expanding family of RAS GTPase-activating proteins (GAPs) that includes mammalian GAP, the yeast Saccharomyces cerevisiae IRA proteins, and the product of the human neurofibromatosis locus, NF1 sar1, like these other proteins, can complement the loss of IRA function in S. cerevisiae. Computer analysis shows that the highest degree of sequence conservation is restricted to a very small number of diagnostic residues represented by the motif Phe-Leu-Arg-X-X-X-Pro-Ala-X-X-X-Pro. We find no evidence that sar1 is required for the effector function of ras1.     

Biochemical specificity of H-2M3a. Stereospecificity and space-filling requirements at position 1 maintain N-formyl peptide binding.

The maternally transmitted Ag is a cell surface product of three gene products: 1) H-2M3a (formerly Hmta), a class I MHC heavy chain; 2) beta 2-microglobulin; and 3) maternally transmitted factor (Mtf), the N-terminus of the mitochondrially encoded ND1 subunit of the reduced form of nicotinamide-adenine dinucleotide dehydrogenase. This class I molecule has been shown to be an N-formyl peptide receptor. Although the N-formyl moiety is necessary for binding to M3a, it is not sufficient. We proposed that the R group of the amino acid in position 1 plays a pivotal role in peptide binding to M3a. To test this hypothesis, analogues differing in size and stereospecificity of the R group were synthesized. Substitutions with other hydrophobic amino acids such as N-formyl phenylalanine and N-formyl valine had no significant effect on the ability of these Mtf alpha analogues to sensitize target cells (M3a, Mtf beta) to M3a, Mtf alpha-specific CTL. In contrast, the nonsubstituted, N-formylated, and N-acetylated glycyl analogues of Mtf beta bound equivalently to M3a in a peptide competition assay. Moreover, the alanine analogue bound in an N-formyl-dependent manner. To determine the limitations of the putative N-formyl pocket, peptide analogues were constructed incorporating D-isomer amino acids. When formylated D-alanine or D-methionine replaced the native methionine, these peptide derivatives did not show significant binding to M3a. Therefore, the presence of a space-filling R group (greater than hydrogen) is necessary for an antigenic peptide to bind M3a in an N-formyl-dependent manner. Additionally, the ability of M3a to discriminate between the optical forms of methionine and alanine demonstrates that this N-formyl pocket is stereospecific in its ability to bind peptide. Thus, we have defined three requirements for peptide binding to M3a: an N-formyl moiety at the amino terminus of the peptide, a space-filling R group at position 1 to maintain this N-formyl specificity, and the correct stereoisomer of the first amino acid.     

Oxidized low-density lipoprotein increases cultured human endothelial cell tissue factor activity and reduces protein C activation

Increasing evidence suggests that the formation of oxidized low-density lipoprotein (Ox-LDL) in vivo is associated with the development of atherosclerotic vascular disease. We investigated the effects of Ox-LDL on two vascular endothelial cell coagulant properties, tissue factor expression, and protein C activation. The Ox-LDL increased human arterial and venous endothelial cell tissue factor activity, with 100 micrograms/ml of Ox-LDL increasing factor activity fourfold. Native LDL modified by incubation with cultured human arterial and venous endothelial cells also induced endothelial cell tissue factor activity. This modification was blocked by coincubation with the antioxidants, probucol or ascorbic acid. It was determined, based on inhibition by known scavenger receptor antagonists (fucoidin, dextran sulfate), that binding of Ox-LDL via the acetyl LDL (scavenger) receptor was partially responsible for the increase in tissue factor expression. Whereas endothelial cell tissue factor expression was increased by incubation with Ox-LDL, protein C activation was reduced approximately 80% by incubating cultured endothelial cells with Ox-LDL. The effect of Ox-LDL on protein C activation was not inhibited by antagonists to the scavenger receptor. These data indicating that an atherogenic lipoprotein can regulate key vascular coagulant activities provide an additional link between vascular disease and thrombosis.     

Ultrastructural cytology of the cyclic corpus luteum of the cow

Corpora lutea (CL) from cows on day 12 of the oestrous cycle were studied by electron microscopy to investigate whether, and how, different subpopulations of luteal cells can be identified in tissue sections. Tissues from 6 CL were examined, and nucleated profiles of luteal cells were classified as large, medium or small on the basis of their areas in electron micrographs. Cut-off points for area categories for large, medium and small-sized cells were based on diameters of greater than 25, 20-25 and less than 20 microns, respectively, if the measured areas were converted to a circular shape after correction for shrinkage. The only qualitative features which distinguished cells of large size from those of small size were the presence of clusters of secretory granules, and of exocytosis of these granules, in large cells only. However, these features were observed in only 59% of large cells, probably primarily due to sampling limitations in single sections. Other qualitative features which have been regarded as diagnostic of large or small luteal cells were observed in cells in all size categories. It was concluded that large and small luteal cells in the cyclic CL of the cow are distinguishable by their ultrastructural features. However, these data do not support the recent suggestion that the mid-cycle CL of the cow contain two subpopulations of large luteal cells in approximately equal numbers.     

Diversity among strains causing bacterial kidney disease in salmonid fish

A study was made of the biochemical, cultural, morphological, physiological and serological characters of 25 Gram-positive bacterial isolates of bacterial kidney disease in salmonid fish. Two distinct homogenous phena and seven single-member clusters were defined as a result of overall similarity based on analyses with the Jaccard coefficient. One phenon was equated withCorynebacterium pyogenes, but the second represents a novel taxon.