Sporulation of Clostridium cylindrosporum on a Defined, Low-Manganese Medium

Clostridium cylindrosporum HC-1 grew and sporulated well on a defined medium. This is the first demonstration of sporulation of a purinolytic clostridium on a defined medium; manganese levels were below those considered essential for sporulation of most Bacillus species. Sporulation appeared to be initiated before exhaustion of the purine substrate.     

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.     

The dependence of glyceroglycolipid orientation and dynamics on head-group structure

The head-group orientations and molecular dynamics of three glyceroglycolipids in aqueous dispersions, as determined by 2H-NMR, are compared. 1,2-Di-O-tetradecyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (alpha-DTGL) and 1,2-di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML), selectively 2H-labelled on the pyranose ring, at the exocyclic hydroxymethyl group, and at C3 of glycerol, have been studied by 2H-NMR and the results compared with those reported earlier for 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL). The alpha-glucolipid exhibits a gel-to-liquid crystal phase transition and a lamellar to hexagonal mesophase transition at temperatures which are similar to those of the beta-anomer, beta-DTGL. However, alpha-DTGL exhibits head group orientations and molecular ordering in the lamellar and hexagonal phases which differ strikingly with those reported for the corresponding beta-glucolipid. Whereas the head group of beta-DTGL is extended away from the bilayer surface into the aqueous phase, that of alpha-DTGL is almost parallel to the bilayer surface. alpha-DTGL exhibits a molecular order parameter of 0.56 which is substantially greater than that of its anomer, beta-DTGL, 0.45. The latter indicates that the head group region of the alpha-glyceroglucolipid is characterized by smaller angular fluctuations than that of beta-DTGL. On entering the hexagonal mesophase the pyranose ring of the beta-glucolipid undergoes a large reorientation relative to the motional axis of the head group, whereas the alpha-anomer exhibits only a small orientational change. 1,2-Di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML) undergoes a phase transition at 47 degrees C, attributed to the unusual lamellar gel to hexagonal phase transition. The pyranose ring of alpha-DTML, in a mixture with dimyristoylphosphatidylcholine (1:9 mol ratio) to give a lamellar liquid crystalline phase, is oriented away from the bilayer surface into the aqueous environment and has an Smol of 0.75. The results for alpha-DTML, 2H-labelled at the C3 position of glycerol, suggest that this segment also has high molecular ordering. alpha-DTML in a lamellar environment has the least flexible membrane surface of the glyceroglycolipids investigated to date. 2H-NMR spin lattice relaxation times have been used to probe the head group motions of the glycolipids. The results indicate that the rate of head group motion increases in the order alpha-DTML less than alpha-DTGL less than beta-DTGL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of and responsiveness to interleukin 2 in autoimmune BXSB mice

BXSB male mice serve as one of several murine models of human systemic lupus erythematosus. T-cell abnormalities in these mice involve decreased production of and responsiveness interleukin 2 (IL-2) and are age-related. The studies presented here investigated the mechanism of these T-cell defects. The results suggest that excessive suppressor-T-cell activity as well as soluble inhibitors of IL-2 production and activity, including PGE, are not responsible for the low levels of IL-2 observed in culture supernatants of Con A-stimulated lymphocytes from "old" (3-6 months) BXSB male mice. Supplementation of Con A-stimulated lymphocyte cultures from BXSB male mice with human IL-1 or normal murine accessory cells did not augment IL-2 production. Reduced proliferative responses were observed in bulk cultures of Con A- or alloantigen-stimulated "old" BXSB male lymphocytes, which were not enhanced by exogenous IL-2. Limiting dilution analysis revealed reduced frequencies of Con A- and alloantigen-inducible IL-2-reactive T cells in these mice. These results suggest intrinsic defects in the ability of T cells from "old" BXSB male mice to be activated to produce and respond to IL-2.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Thyroid hormone modulation of TRH precursor levels in rat hypothalamus, pituitary, thyroid and blood

In the present study we have examined the in vivo effects of thyroid hormones and TRH on tissue and blood levels of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Using specific radioimmunoassays (RIAs), we measured TRH immunoreactivity (TRH-IR) and TRH-Gly-IR concentrations in blood, hypothalamus, anterior and posterior pituitary, and thyroid in euthyroid, hypothyroid and thyroxine (T4)-treated 250 g male Sprague-Dawley rats. TRH-Gly-IR and TRH-IR were detected in all of these tissues. Highly significant positive correlations between whole blood TRH-Gly-IR levels and the corresponding serum TSH values (p less than 0.01), whole blood TRH-IR versus serum TSH (p less than 0.01) and whole blood TRH-Gly-IR versus whole blood TRH-IR (p less than 0.01) are consistent with cosecretion of TRH and TRH precursor peptides into the circulation. Euthyroid rats injected with TRH IP (1 microgram/100 g b.wt.) and hypothyroid rats had 4-fold higher whole blood TRH-Gly-IR levels compared to euthyroid controls (p less than 0.0005). Injection of TRH into euthyroid rats significantly increased the TRH-Gly-IR concentration in the hypothalamus, anterior and posterior pituitary and thyroid. The increase in blood TRH-Gly-IR following intravenous TRH may be due, in part, to partial saturation of TRH-degrading enzymes in blood and cell membranes. The ratio of TRH-Gly to TRH was significantly increased in the anterior pituitary by hypothyroidism and TRH injection, suggesting that thyroid hormones and TRH regulate the alpha-amidation of TRH-Gly to form TRH in this tissue. TRH-Gly levels of pooled pituitary and thyroid extracts quantitated by a combination of TRH-Gly RIA and high performance liquid chromatography (HPLC) revealed several-fold increases following incubation at 60 degrees C. Heating at this temperature may block the alpha-amidation activity in extra-hypothalamic tissues but not the "trypsin-like" enzymes which cleave prepro-TRH into TRH-Gly-immunoreactive peptides.