Cyclic adenosine-3',5'-monophosphate stimulates the acute release of placental lactogen from human trophoblast cells

cAMP has been shown to be a second messenger in the release of many hormones and other secretory products. To determine whether cAMP also plays a role in the mechanism of release of human placental lactogen (hPL), we examined the effects of (Bu)2cAMP, isobutyl methylxanthine, forskolin, and cholera toxin on the acute release of hPL from an enriched fraction of hPL-producing trophoblast cells. Static cultures of trophoblast cells exposed to (Bu)2cAMP (5 mM) for 2 h released 2.6 times as much hPL as control cells (P less than 0.01) during the first 0.5 h of exposure. The increase in hPL release was followed by a decrease rate of release during the subsequent 1.5 h. Perifused trophoblast cells (1.5 X 10(6) exposed to 5 mM cAMP for 20 min released 3.2 times as much hPL as control cells. The rate of hPL increased markedly during the first 10 min of exposure, rapidly decreased toward control values during the remainder of the exposure period, and then declined to a subnormal rate for the next 30 min before returning to normal to control values. (Bu)2cAMP, however, had no acute effects on the release of human CG or the release of the cytosolic enzymes alkaline phosphatase and lactic dehydrogenase. The phosphodiesterase inhibitors theophylline (5 mM) and isobutyl methylxanthine (0.5 mM) and the adenylate cyclase activators forskolin (5 micrograms/ml) and cholera toxin (25 micrograms/ml) stimulated hPL release by 75-95%. These results strongly suggest that cAMP is a second messenger in the acute release of hPL.     

Demonstration that monocytes rather than lymphocytes are the insulin-binding cells in preparations of humah peripheral blood mononuclear leukocytes: implications for studies of insulin-resistant states in man.

Insulin receptors have been demonstrated on mononuclear leukocytes prepared by centrifugation of buffy coats from normal blood donors on Ficoll-Hypaque gradients. The cell type that specifically binds insulin in this mixture of lymphocytes and monocytes has never been clearly identified, although it was assumed to be the lymphocyte since this cell constitutes about 80% of the population. In the present studies, insulin-binding assays were performed on the mononuclear leukocyte preparation before and after selective depletion or enrichment for monocytes using glass wool or Sephadex G-10 adherence columns. The amount of 125-I-labeled insulin specifically bound correlated significantly with the number of monocytes but not with the number of B or T lymphocytes. Approximately 90% of the specific insulin binding of this preparation could be accounted for by its content of monocytes. The amount of binding was unaffected by phagocytosis of latex particles or by metabolic inhibitors added to prevent endocytosis. Autoradiograms made on smears of whole peripheral blood and mononuclear leukocytes demonstrated that all of the cells that bound 125-I-labeled insulin were large mononulcear cells, 85-90% of which could be identified as monocytes by morphological criteria or by the functional criterion of latex particle ingestion. Since insulin receptor concentration may be altered in disease states in man, it is essential, when using this cell population for detecting such changes, to quantitate the number of monocytes in the preparation so that the insulin-binding data can be appropriately interpreted.     

Relaxin stimulates the synthesis and release of prorenin from human decidual cells: evidence for autocrine/paracrine regulation

Porcine relaxin caused a time- and concentration-dependent increase in the release of renin from decidual cells cultured over a 96 h period. The increase in renin release occurred 24-48 h after exposure and was maximal at 48-72 h. Half-maximal stimulation occurred at a relaxin concentration of 5 ng/ml, and maximal stimulation (250-270%) occurred at concentrations greater than or equal to 10 ng/ml. At each time, greater than 95% of the renin released into the medium was in the form of prorenin. The stimulation of renin release was paralleled by a stimulation of cellular renin content and was completely inhibited by cycloheximide, indicating that relaxin also stimulated renin synthesis. Since renin is present in both cytotrophoblast and decidual cells, these results suggest a paracrine and/or autocrine relationship between relaxin- and prorenin-secreting cells.     

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.     

Stimulation of ovine placental lactogen secretion by arachidonic acid

To determine whether arachidonic acid stimulates the secretion of ovine placental lactogen (oPL), arachidonic acid was infused as an intravenous bolus into pregnant ewes and fetuses. Plasma oPL concentrations were determined in mothers and fetuses before and for 5 h after infusion. The administration of 12.5 mg arachidonic acid (0.15-0.2 mg/kg, n = 11 experiments) to the pregnant ewes caused an increase in maternal plasma oPL concentrations of 73.9 +/- 15.6% (S.E.M.) and 60.8 +/- 18.1% above the pretreatment concentrations at 4 and 5 h respectively (P less than 0.01 in each instance). The infusion of 25 mg arachidonic acid (n = 8) caused increases of 96.0 +/- 19.1% and 100.3 +/- 26.4% (P less than 0.005), and the stimulation was not inhibited by the cyclo-oxygenase inhibitors indomethacin and ibuprofen. In contrast to arachidonic acid, vehicle alone or palmitic acid had no effects on plasma oPL concentrations. Despite the increase in maternal plasma oPL concentrations, plasma oPL concentrations in the fetus remained unchanged after the maternal infusions. The infusion of arachidonic acid (0.5-1.5 mg/kg) directly into six fetuses had no effects on either fetal or maternal oPL concentrations. These studies indicate that arachidonic acid stimulates maternal plasma oPL concentrations but has no effect on fetal oPL concentrations and the stimulation of oPL secretion is not due to the conversion of arachidonic acid to prostaglandins or other cyclo-oxygenase products.     

Immune and autoimmune aspects of diabetes mellitus

The immune and autoimmune aspects of diabetes mellitus are reviewed. Emphasis is given to the clinical association of diabetes with other autoimmune disease; the increased incidence of organ-specific autoimmunity in diabetic patients; the occurrence of humoral and cell-mediated antipancreas (islet) autoimmunity in diabetes; the association of HLA with juvenile-onset, insulin-dependent diabetes mellitus and with certain specific subpopulations of diabetic patients; the possible role of viruses in the etiology of diabetes; and the occurrence of alterations in humoral and cell-mediated immunity, granulocyte function, and the host defense against infectious agents in human diabetics and in animals with experimental diabetes.     

Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the beta-lactam binding site

There are several major differences between the penicillin-binding proteins (PBPs) of highly penicillin-resistant and -susceptible strains of pneumococci. The highest-molecular-size PBP 1a (98 kilodaltons [kDa]) of susceptible pneumococci is not detectable in resistant bacteria. Instead, resistant strains contain a PBP of smaller size: 92 and 94 kDa in South African strains 8249 and A95210, respectively, and 96 kDa in New Guinea strain 2955 (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). Using antibodies prepared against PBP 1a of penicillin-susceptible pneumococci, we demonstrated that these anomalous-sized proteins in the resistant strains are immunologically related to PBP 1a of penicillin-susceptible bacteria. A second difference between the PBP patterns of strain 8249 and the susceptible pneumococci is that the 78-kDa PBP 2b is not detectable by the radioactive penicillin binding assay in the resistant strain. Using antibodies prepared against PBP 2b of susceptible cells, we demonstrated the presence of PBP 2b in membrane preparations from strain 8249 cells. Thus, the poor detection of this PBP appears to be related to its greatly decreased affinity for the antibiotic molecule. We also compared the patterns of penicillin-labeled peptides derived from PBPs of resistant and susceptible cells during partial proteolysis by V8 protease. Several changes were observable in small peptides carrying the beta-lactam binding site generated from the high Mr (PBP 1a-related) binding proteins. In contrast, no differences in the pattern of penicillin-labeled peptides were seen when the pattern of PBP 2a of susceptible pneumococci was compared with the peptide pattern of PBP 2a from resistant strains. One of the resistant isolates (strain 2955) also had a PBP 3 with a higher-than-normal molecular weight. This protein gave strong positive reaction with antibodies against PBP 3 of susceptible cells. Examination of the pattern of penicilloyl peptides generated from the susceptible and resistant PBP 3s during partial proteolysis revealed only differences which seem to reside distant from the beta-lactam binding site.     

Alterations in immunological reactivity in encephalomyocarditis virus-induced murine diabetes. I. Defective primary IgM plaque forming cell responses to sheep erythrocytes: correction by islet cell transplantation.

Increasing data suggest a possible viral aetiology of juvenile onset, insulin-dependent diabetes mellitus. The M variant of the encephalomyocarditis (EMC) virus infects murine pancreatic beta cells and causes a diabetes like syndrome in susceptible strains of mice. Abnormalities in immunological function have been documented in patients with diabetes mellitus and in spontaneous, streptozotocin-induced and alloxan-induced diabetes in animals. The present study documents a significant impairment of the ability of mice with EMC virus (M variant)-induced diabetes to generate a direct, IgM PFC response after in vivo immunization with sheep erythrocytes. This abnormality appears to be a direct consequence of the diabetic state and not EMC virus infection, per se, since mice infected with EMC virus that do not become diabetic have normal direct PFC responses and islet cell transplantation, which cures the diabetes, corrects the defect in PFC responsiveness.