Toxic shock syndrome toxin-1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells

Toxic shock syndrome toxin-1 (TSST-1) is a 22-kDa exotoxin produced by most Staphylococcus aureus strains responsible for toxic shock syndrome. TSST-1 is a mitogen for human T cells. The mechanism of T cell activation by TSST-1 was investigated. TSST-1 induced IL-2R expression, IL-2 synthesis, and proliferation in T cells in a monocyte-dependent fashion. Neither IL-1 nor IL-2, alone or in combination, substituted for monocytes in supporting TSST-1-induced mitogenesis. We investigated the mechanism by which TSST-1 induces initogenesis. TSST-1 failed to induce ADP-ribosylation of T cell membrane proteins. However, the toxin induced transient translocation of protein kinase C from cytosol to plasma membranes and also induced the mobilization of cellular Ca2+ stores in both PBMC and the Jurkat human tumor T cell line, suggesting that TSST-1 triggered inositol phospholipid turnover. This was directly demonstrated to be the case in both cellular preparations studied. TSST-1 induced the increased synthesis of the inositol phospholipid phosphatidyl inositol, phosphatidyl inositol-4 phosphate, and phosphoinositol inositol-4,5-bisphosphate, and induced the breakdown of inositol phospholipid as evidence by the accumulation of phosphatidic acid and inositol phosphates. We conclude that the action of TSST-1 involves the induction of inositol phospholipid turnover, protein kinase C activation, and mobilization of cellular Ca2+ stores. This effect is similar to that of mitogenic lectins and of anti-CD3 antibodies.     

A comparison of the lipid-lowering and intestinal morphological effects of cholestyramine, chitosan, and oat gum in rats

Cholestyramine, chitosan, and oat gum are lipid-lowering compounds. Cholestyramine use in humans may contribute to colonic adenocarcinoma; chitosan and oat gum are being studied in the rat to determine their potential for human use. To compare these compounds, we fed three groups of 10 male Sprague-Dawley rats one of the substances at 5% of diet with 1% cholesterol and 0.2% cholic acid; two other groups were fed cellulose with and without 1% cholesterol and 0.2% cholic acid. All groups had similar food intake and weight gains. Cholesterol feeding increased total liver lipids almost 3-fold and liver cholesterol concentration almost 10-fold. Cholestyramine, oat gum, and chitosan all significantly lowered liver cholesterol with cholestyramine feeding yielding levels identical to the noncholesterol-fed basal group. Chitosan and oat gum lowered liver cholesterol moderately. Cholestyramine and chitosan both significantly lowered serum cholesterol compared to the cellulose group. Oat gum was less effective. Hemoglobin and serum iron were similar in all groups except the oat gum group, which had decreased serum iron. Histological examination of small and large bowel with morphometry revealed statistically significant increases in both proximal and distal small bowel and distal large bowel mucosal thickness in the cholestyramine-fed group. No changes were noted in the proximal large bowel. Neither chitosan nor oat gum produced mucosal change other than an increase in the distal small bowel with the oat gum diet. Chitosan may have lipid-lowering effects similar to those of cholestyramine without the deleterious changes in intestinal mucosa.     

Treatment of canine aspiration pneumonitis: fluid volume reduction vs. fluid volume expansion

The aspiration of gastric acid causes pulmonary edema and hypoxemia. One approach to the management of this syndrome is to raise cardiac output (Qt) and O2 delivery (QO2) to ensure tissue oxygenation (VO2) at the risk of increasing the edema. Another approach reduces the edema by reducing pulmonary microvascular pressure (Pmv) at the risk of reducing QO2 and VO2. We compared these approaches in 24 anesthetized, ventilated dogs with pulmonary wedge pressure (Ppw), a clinical approximation of Pmv, of 12.5 mmHg. Before and again 1 h after endobronchial instillation of 0.1 N HCl, we measured Qt, QO2, VO2, venous admixture, and in vivo extravascular lung liquid. The dogs were then randomly divided into four equal groups: 1) 12.5 mmHg Ppw, high Qt; 2) 7.5 mmHg Ppw, intermediate Qt; 3) 4.5 mmHg Ppw, low Qt; and 4) 4.5 mmHg Ppw plus dopamine, intermediate Qt. Measured values were followed for 4 more h, after which the lungs were excised to compare wet weight-to-body weight ratios (W/B). When plasmapheresis reduced Ppw at 1 h, edema did not increase further and W/B of groups 2 (21 +/- 3), 3 (18 +/- 3), and 4 (22 +/- 3) were significantly less than in group 1 (27 +/- 3) (P less than 0.001). Although Qt decreased with Ppw, increased hematocrit and reduced venous admixture maintained QO2 in group 2 but not in group 3. In group 4 an intermediate Qt maintained QO2 even at 4.5 mmHg Ppw but edema increased to the group 2 level presumably because Pmv rose with Qt on dopamine. VO2 remained constant over time in each group. These data demonstrate that canine HCl-induced pulmonary edema, measured in vivo or gravimetrically, is very sensitive to reductions in Pmv. Moreover, the lowest Pmv (and QO2) was well tolerated because an O2 supply dependency of VO2 was not observed.     

Pressure, flow, and density relationships in airway models during constant-flow ventilation

Adequate CO2 elimination and normal arterial PCO2 levels can be maintained in dogs during apnea by delivering a continuous flow of inspired gas at high flow rate (1-3 l.min-1.kg-1) through tubes placed in the main-stem bronchi. However, during constant-flow ventilation (CFV) the mean alveolar pressure is increased, causing increased lung volume despite low pressures in the trachea. We hypothesized that the increased dynamic alveolar pressures during CFV were due to momentum transfer from the high-velocity jet stream to resident gas in the lung. To test this, we simulated CFV in straight tubes and in a branched airway model to determine whether changes in gas flow rate (V), gas density (rho), and tube diameter (D) altered the pressure difference (delta P) between alveoli and airway opening in a manner consistent with that predicted by conservation of momentum. Momentum analysis predicts that delta P should vary with V2, whereas measurements yielded a dependence of V1.69 in branched tubes and V1.9 in straight tubes. Substitution of heliox (80% He-20% O2) for air significantly reduced lung hyperinflation during CFV. As predicted by momentum transfer, delta P varied with rho 1.0. Momentum analysis also predicts that delta P should vary with D-2.0, whereas measurements indicated a dependence on D-2.02. The influence of V and rho on depth of penetration of the jet down the airway was explored in a straight tube model by varying the flow rate and gas used. The influence of geometry on penetration was measured by changing the ratio of jet-to-airway tube diameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase

The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.     

Structure and evolution of insulins: implications for receptor binding.

Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common 'insulin fold' is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

3-phenylacetylamino-2,6-piperidinedione inhibition of rat Nb2 lymphoma cell mitogenesis

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1-12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P less than 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.     

Do plasma ACTH and cortisol concentrations in the late-gestation fetal sheep vary diurnally?

This study tested the hypothesis that fetal plasma ACTH and cortisol concentrations vary diurnally, and the mean concentration and the amplitude of the rhythm vary as a function of fetal gestational age. Nine chronically-catheterized fetal sheep were studied between 120 and 142 days' gestation. All of the fetuses were born spontaneously and alive. The pregnant ewes were maintained in a room with a regular light cycle (on at 07.30, off at 17.30). Food and water were available ad libitum. Blood samples were drawn at 4-h intervals throughout a 24-h period. There were no significant daily variations in fetal plasma ACTH, cortisol, or progesterone concentrations, except in the last 3 days of fetal life. In these fetuses ACTH and cortisol concentrations were increased in the afternoon and evening. We conclude that there is no diurnal rhythm in ovine fetal hypothalamo-pituitary-adrenal axis activity, and that the increased plasma concentrations of ACTH and cortisol in the afternoon and evening hours of the last few days of fetal life might be a response to increased uterine contraction activity.