Immunoreactive and biologically active somatostatin in human and sheep milk

The presence of immunoreactive and biologically active somatostatin in sheep and human milk has been demonstrated. Milk somatostatin exhibits similar chromatographic behavior to that of synthetic somatostatin-14 on both reversed-phase C18 and cation-exchange high-performance liquid chromatography columns. Milk, in contrast to plasma, contains only somatostatin-14-like material. Milk somatostatin was capable of inhibiting the basal and the prostaglandin-induced release of growth hormone from anterior pituitary cell cultures in a pattern similar to synthetic somatostatin-14. The concentrations of the peptide, as determined by radioimmunoassay, were found to be 113 pg/ml in human milk and 150 +/- 4.8 pg/ml (mean +/- range) in sheep milk. These values are severalfold higher than the corresponding concentration of the peptide in the plasma of these species. These findings are analogous to our previous observations concerning two other hypothalamic hormones, luliberin and thyroliberin [Baram, T., Koch, Y., Hazum, E. and Fridkin, M. (1977) Science (Wash. DC) 198, 300-302]. The high concentration of somatostatin and other neuropeptides in milk implies either an active concentrating mechanism in the mammary gland or an additional extrahypothalamic source for the synthesis and release of these peptides.     

Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA

Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

Ultrastructural localization of the calcium-binding protein parvalbumin in neurons of the song system of the zebra finch,Poephila guttata

Summary The distribution of parvalbumin (PV) within neurons of the vocal motor nucleus hyperstriatum ventralepars caudalis (HVc) was investigated in the forebrain of adult male zebra finches by means of light and electron microscopy using the indirect immunoperoxidase technique. Parvalbumin-reaction product was located in the amorphous material of perikarya, dendrites and nuclei, and associated to microtubuli, postsynaptic densities and intracellular membranes; it was found in some axons and Gray type-2 boutons, but rarely in type-1 boutons and never in the Golgi apparatus. These observations suggest that parvalbumin may regulate calcium-dependent processes at the postsynaptic membrane and in the cytosol. Furthermore, the partial association of parvalbumin to microtubuli points to an involvement in calcium-dependent tubular functions. Calcium currents and microtubular assembly or transport may be relevant for the known functions of HVc in song learning.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.     

Composition of Caulobacter crescentus murein synthesized in the presence of glycine

Abstract Glycine added to the growth medium of Caulobacter crescentus was found to substitute Cterminal alanine in the peptide side chains of the murein of this species. Murein synthesized in vivo and in vitro in the presence of glycerine was poorly crosslinked as was new murein formed in the presence of the amino acid. The reduced cross-linkage seems to be due to the effect of glycine on the formation of trimeric muropeptides as revealed by high-performance liquid chromatography (HPLC) muropeptide analysis of murein formed in the presence and absence of the amino acid.     

The disappearance of injected prostaglandins from the circulation of adult female australian field crickets,Teleogryllus commodus

During the first 2 h following injection of 0.02 μCi of [¹⁴C]-prostaglandin E₂ into the abdomen of adult virgin female crickets, T. commodus, the concentration of radioactivity in the circulating hemolymph decreases. The reduction is associated with the increase in radioactivity in the Malpighian tubule/hindgut complex, ovaries, fat body, and, to a much smaller extent, ventral nerve cord and flight muscles. The finding that most, but not all, of the radioactivity in the hindgut is located in the contents of the lumen suggests that a high proportion of prostaglandins circulating in the hemolymph of T. commodus is eliminated by the usual excretory pathway. We suggest that the differential uptake of label from the circulating hemolymph by various tissues may be related to possible physiological functions that remain to be discovered.     

Regulation of the β-ketoadipate pathway in Rhizobium japonicum and bacteroids by succinate

Rhizobium japonicum 61-A-101 and its bacteroids catabolize phenol and p-hydroxybenzoate. With phenol as a carbon source, utilization started only after a prolonged lag phase while p-hydroxybenzoate was almost instantancously metabolized. Succinate, which supports rapid growth of Rhizobium japonicum, completely repressed respication of phenol; the oxidation of p-hydroxybenzoate was partially inhibited. Pyruvate, supporting slower growth than succinate, retarded the onset of phenol consumption but did not affect its maximum rate.Catabolite repression of phenol utilization by succinate appears to be a characteristic feature of rhizobia. In Pseudomonas putida which also actively metabolizes phenol, succinate had no effect on phenol utilization.     

Bluelight-induced,flavin-mediated transport of redox equivalents across artificial bilayer membranes

Summary This paper continues our studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochromec, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e ^–(EDTA)-and 2e ^–(BH₃CN^–) donors across the membrane finally reducing the trapped cytochromec both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented.