Guanine nucleotide-dependent, pertussis toxin-insensitive regulation of phosphoinositide turnover by bradykinin in bovine pulmonary artery endothelial cells

In this paper we examine the effect of the vasodilator peptide bradykinin on endothelial cell regulation of phosphoinositide (PI) turnover. The data show that the activation of PI turnover by bradykinin in bovine pulmonary artery endothelial cells is insensitive to pertussis toxin, which ADP ribosylates a membrane protein of mol wt 40,000. However, this effect of bradykinin can be potentiated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S), an activator of G proteins, and depressed by guanosine 5'-O-(2-thio)diphosphate (GDP beta S), an inhibitor of G proteins. After endothelial cells were preincubated for 1 h with GTP gamma S, there was a three- to fourfold increase in PI turnover. Preincubation of cells with GDP beta S did not affect the basal level of PI turnover, but completely prevented activation of PI turnover by bradykinin. 4 beta-Phorbol-12 beta-myristate-13 alpha-acetate can block the bradykinin-stimulated inositol monophosphate formation in cultured endothelial cells. The effects of bradykinin on PI turnover were blocked by B2 antagonists but not by B1 antagonists. Taken together, these results indicate that in endothelial cells the bradykinin B2 receptor is coupled to phospholipase C via a G protein (or proteins) that is not a substrate for pertussis toxin (neither Gi nor Go).     

Maternal morbidity associated with in utero transfer.

OBJECTIVE--To determine the extent of maternal morbidity associated with in utero transfer. DESIGN--Retrospective study of 190 consecutive cases over two years. SETTING--Liverpool Maternity Hospital. PATIENTS--190 Pregnant women were transferred to the hospital under the in utero transfer arrangements from district general hospitals both within and outside the Mersey region. The women admitted were divided into two categories: those in threatened or established uncomplicated preterm labour and those who may or may not have been in threatened or established preterm labour but who had coexisting complicating factors affecting the mother or fetus, or both. INTERVENTIONS--Planned delivery of the fetus if indicated and arrangements for appropriate postpartum care of the mother. MAIN OUTCOME MEASURE--Assessment of the progress of labour and, if appropriate, resuscitation of the mother. RESULTS--Women who were transferred with no coexisting disease (124) had relatively uncomplicated deliveries whereas those transferred with coexisting diseases (66) exhibited considerable morbidity and 17 of these required prolonged intensive monitoring after delivery. CONCLUSIONS--In utero transfer in healthy mothers may have benefits for babies born very prematurely. If mothers have coexisting disease, however, the desirability of transfer should be reviewed urgently in the light of the considerable maternal morbidity associated with these problems. In these cases transfer may introduce an additional hazard.     

Oxidative metabolism of energy substrates by preimplantation mouse embryos in the presence of platelet-activating factor

The effects of exogenous platelet-activating factor (PAF, 0.0186-18.6 microM) on the production of CO2 from [U-14C]glucose and [1-14C]lactate by mouse embryos in vitro were investigated. Two-cell embryos displayed significant dose-dependent responses for both energy substrates. The maximal response was observed at 9.3 microM-PAF for glucose metabolism and 1.86 microM-PAF for lactate with increases of 62% and 18%, respectively, over control treatments. After culture from the 2-cell stage for 72 h in the presence of PAF, the resulting blastocysts exhibited a significant dose-dependent increase in metabolism of lactate. It was also apparent that such embryos were not desensitized to PAF as demonstrated by a further enhancement of the metabolic response after re-exposure to PAF. The specificity of action of PAF was confirmed by the absence of any effect on the oxidative metabolism of glucose by lyso-PAF (a catabolite of PAF) over a concentration range of 0.0202-20.2 microM and by the demonstration that SRI 63-441 (a PAF-receptor antagonist) significantly reduced the amount of CO2 produced from glucose in response to 9.3 microM-PAF and abolished the effect on lactate metabolism in response to 1.86 microM-PAF. These results demonstrate a specific, direct influence of exogenous PAF on the oxidative metabolism of glucose and lactate by the preimplantation mouse embryo and suggest an autocrine role for embryo-derived PAF in early pregnancy.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Adrenal glucocorticoids regulate adipsin gene expression in genetically obese mice

Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.     

A 14-kilodalton selenium-binding protein in mouse liver is fatty acid-binding protein

In a previous study, we purified three selenium-binding proteins (molecular masses 56, 14, and 12 kDa) from mouse liver using column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aim of the present study was to determine the amino acid sequence of the 14-kDa protein thereby establishing any relationship with known proteins. Although the amino terminus of the 14-kDa protein was blocked, separate in situ digestions of the protein with endoproteinases Glu-c and Lys-c gave overlapping peptides that provided a continuous sequence of 93 amino acids. This sequence exhibited a 92.5% sequence homology with rat liver fatty acid-binding protein. In situ enzymatic digestion and partial sequencing of a 12-kDa selenium-binding protein revealed identical homology to the 14-kDa protein. The 14-kDa protein bound specifically to an oleate-affinity column from which the protein and 75Se coeluted. Delipidation or sodium dodecyl sulfate treatment failed to remove 75Se from the protein, indicating that the selenium moiety was tightly bound to the protein. These observations confirm that the mouse liver selenium-binding 14-kDa protein is a fatty acid-binding protein. The nature of the selenium linkage to the protein still needs to be explored.     

The cGMP-gated channel of bovine rod photoreceptors is localized exclusively in the plasma membrane

Although there have been several reports pertaining to the existence of the cGMP-gated channel in the disk membrane of rod photoreceptors, its density there relative to that of the photoreceptor plasma membrane is unknown. Using immunoblotting, immunohistochemical, and reconstitution techniques on purified disk and plasma membrane preparations, we found that the density of channels in the plasma membrane was at least 50-fold higher than that of the disk membrane. Purification of membrane fractions without prior digestion of cytoskeletal components by mild trypsinization was found to increase the amount of channel protein present in disk membrane preparations. We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptor cell.     

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.