Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores

Abstract Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and were tentatively identified as Chromatium purpuratum . Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of intracellular localization of antibiotic activity in a marine bacterium.     

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.     

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.     

The use of the rat hepatoma cell line MH1C1 to investigate the stimulus-secretion response of hepatic acute phase proteins

The rat hepatoma cell line MH1C1 has been characterized to show a stimulated secretion of C-reactive protein in response to both leukocyte supernatant and a purified human interleukin-1 preparation. The time-dependency and dose-response relationship of CRP secretion were comparable to and somewhat more sensitive than the effects of leukocyte supernatant and purified human interleukin-1 on the proliferative rate of murine thymocytes; the proliferative rate of the hepatoma cell line MH1C1 was unchanged under these conditions. Agents which affect the thymocyte bioassay response to interleukin-1 namely interleukin-2, lipopolysaccharide, concanavalin A and phytohemagglutinin showed no effect on the C-reactive protein release of the MH1C1 cell line. These data strongly support the suitability of this cell line for the in vitro study of the hepatic acute phase stimulus-secretion response.     

Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells

An inhibitor of plasminogen activator (PA) secreted by a tumorigenic, but non-metastatic, rat mammary adenocarcinoma cell line has been purified to apparent homogeneity and characterized. It strongly inhibited human urokinase, but was 100 times less potent in inhibiting bovine trypsin and had no effect on plasmin or thrombin. A secreted, urokinase-type PA (Mr 48 000) and a cell-associated PA from a metastatic rat adenocarcinoma cell line were also strongly inhibited. In contrast, a tissue-type PA (Mr 66 000), secreted by human melanoma cells, was only slightly inhibited. Purified inhibitor showed a band of Mr 66 000 in sodium dodecyl sulphate/polyacrylamide gel electrophoresis and an isoelectric point of 4.5 after chromatofocusing. The inhibition of human urokinase was non-competitive.     

Characterization of the platelet agglutinating activity of thrombospondin

Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.     

Biochemical genetic stock structure of the southern African anchovy, Engraulis capensis Gilchrist

The geographical distributions of inherited biochemical markers were used to measure the amount of genetic isolation between stocks of Namibian and South African anchovy, Engraulis capensis . A contingency-table analysis of allele frequencies for 10 polymorphic protein-coding loci revealed no significant frequency differences between spawning areas. The average Nei genetic distance between samples was 0.0003 and there were no geographic trends in the amount of genetic distance between populations. Average population heterozygosity for 31 loci was 0.115 and this accounted for 99.26% of the total genetic variation. The remaining 0.24% was due to all temporal and spatial differences combined. The observed amount of genetic divergence between populations was used to estimate the amount of migration between spawning areas, using the stepping-stone model of migration. As few as 13 migrants may account for the observed genetic divergence between spawning areas. The validity of using the genetic stock concept in the management of marine fishes is discussed.     

A disulfide-bonded short chain collagen synthesized by degenerative and calcifying zones of bovine growth plate cartilage

Studies of collagen synthesis by specific sections of individual fetal bovine costochondral junction growth plates were conducted and histologically related to the zones from which the sections were derived. Sections were metabolically labeled in organ culture to examine the synthesis of collagen and its precursors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Type II collagen was the major species synthesized in all tissue sections; 1 alpha, 2 alpha, 3 alpha collagen chains were synthesized in all growth plate sections and to a small extent in the fetal structural cartilage. A short chain collagen was synthesized predominantly in the zones of degeneration and provisional calcification and accounted for 8-12% of the radioactivity in this section. This short chain collagen has 63-kDa subunits which are converted to 46-kDa species by limited proteolysis with pepsin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that both the pepsin- and non-pepsin-treated forms of short chain collagen are disulfide-bonded. Digestion with bacterial collagenase showed that the 46-kDa and a major portion of the 63-kDa forms are collagenous. Pulse-chase studies in organ culture did not demonstrate an obvious precursor to the 63-kDa form, and there was no conversion to the 46-kDa after 20 h. Synthesis of short chain collagen appears to be specific to the process of endochondral ossification in the growth plate; its appearance may be critical to this transition process.     

Endocrine and metabolic responses of the collared peccary (Tayassu tajacu) to immobilization with ketamine hydrochloride

Serial physiological responses were examined for 150 min from captive collared peccaries during immobilization with ketamine hydrochloride. Rectal temperatures decreased significantly (P less than 0.01) during anesthesia. Serum concentrations of total proteins, albumin, cholesterol, alanine aminotransferase, and calcium declined significantly (P less than 0.05) during the first 45 min post-immobilization before stabilizing. Concentrations of lactate dehydrogenase and alkaline phosphatase in sera showed similar but nonsignificant (P greater than 0.05) trends. Inorganic phosphorus and aspartate aminotransferase concentrations increased significantly (P less than 0.05) throughout the trial. Concentrations of serum glucose and glucocorticoid during the immobilization period were highly variable between individuals. Serum electrolytes, urea nitrogen, creatinine, gammaglutamyl transferase and progesterone were not significantly (P greater than 0.05) affected by immobilization. Elevations in serum testosterone were noted. Results indicated appropriate sampling times relative to immobilization for assay of particular serum biochemicals and steroid hormones during investigations of the physiology of the collared peccary.     

Leakage from Seedling Roots, Inoculated with Phytophthora cinnamomi

A sequential study of electrolyte leakage from roots inoculated with Phytophthora cinnamomi demonstrated changes in leakage in response to infection. These changes may be characterised in terms of susceptibility and resistance. Field resistant species showed two types of response (i) rapid leakage 2—4 h after inoculation, such as in Eucalyptus calophylla, E. maculata and Gahnia radula, (ii) no significant increase in leakage with infection, for example in cereals and in juncus bufonius. Susceptible species, such as Xanthorrhoea australis, E. sieberi and E. marginata showed slow but continually increasing leakage after inoculation, and usually lost significantly more electrolyte than field resistant species. Both electrolyte leakage and susceptibility of two Eucalyptus species varied with root temperature. Roots of both the susceptible and field resistant Eucalyptus species grown at 14°C showed similar leakage patterns to those grown at 24°C although no lesions formed at 14°C. Both amount of leakage and lesion length increased at 28°C. E. marginata (susceptible) lost significantly greater quantities of electrolyte than E. calophylla at each temperature tested. Leakage from artificially wounded roots did not vary with either temperature or with host susceptibility. No significant changes in conductivity were recorded with saprophytic colonization of roots, or with incubation in either low molecular weight culture filtrate or β-glucan solutions. Vacuum infiltration with the culture filtrate or the β-glucan slightly increased initial electrolyte loss in comparison with that of the controls. The increased leakage from infected roots may be toxin mediated or due to enzymie degradation of host plasma membranes, and occurred within the region of the limited lesion in the case of field resistant species, compared with the larger zone of extending necrosis characteristic of susceptible species.