The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.     

Activity of the natural algicide, cyanobacterin, on eukaryotic microorganisms

Abstract The natural product cyanobacterin has been shown to be toxic to most cyanobacteria at a concentration of approx. 5 μM. We demonstrate here that cyanobacterin will also inhibit the growth of most eukaryotic algae at a similar concentration. Some algae, such as Euglena gracilis , are resistant because they are able to maintain themselves by heterotrophic nutrition. Others, such as Chlamydomonas reinhardtii , can apparently induce a detoxification mechanism to maintain photosynthesis in the presence of low concentrations of the inhibitor. Non-photosynthetic microorganisms are not affected by cyanobacterin.     

The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.     

Survey of metal tolerance in moderately halophilic eubacteria

The tolerance patterns, expressed as MICs, for 250 moderately halophilic eubacteria to 10 heavy metals were surveyed by using an agar dilution method. The moderate halophiles tested included 12 culture collection strains and fresh isolates representative of Deleya halophila (37 strains), Acinetobacter sp. (24 strains), Flavobacterium sp. (28 strains), and 149 moderately halophilic gram-positive cocci included in the genera Marinococcus, Sporosarcina, Micrococcus, and Staphylococcus. On the basis of the MICs, the collection strains showed, overall, similar responses to silver, cobalt, mercury, nickel, lead, and zinc. All were sensitive to silver, mercury, and zinc and tolerant of lead. The response to arsenate, cadmium, chromium, and copper was very heterogeneous. The metal susceptibility levels of the 238 freshly isolated strains were, in general, very heterogeneous among the four taxonomic groups as well as within the strains included in each group. The highest toxicities were found with mercury, silver, and zinc, while arsenate showed the lowest activity. All these strains were tolerant of nickel, lead, and chromium and sensitive to silver and mercury. Acinetobacter sp. strains were the most heavy-metal tolerant, with the majority of them showing tolerance of eight different metal ions. In contrast, Flavobacterium sp. strains were the most metal sensitive. The influence of salinity and yeast extract concentrations of the culture medium on the toxicity of the heavy metals tested for some representative strains was also studied. Lowering the salinity, in general, led to enhanced sensitivity to cadmium and, in some cases, to cobalt and copper. However, increasing the salinity resulted in only a slight decrease in the cadmium, copper, and nickel toxicities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acids bound to vitamin D-binding protein (DBP) from human and bovine sera

Human and bovine vitamin D-binding protein (DBP) have been isolated from serum by a method that does not involve denaturing steps. This method includes Cibacron Blue-Sepharose chromatography, gel filtration, DEAE-Sephadex chromatography and albumin immunoadsorption. Analysis of fatty acids bound to the isolated human and bovine DBP showed molar ratios of fatty acid to protein of 0.4 and 1.3 respectively meanwhile human and bovine albumin have bound 1.8 and 1.5 moles per mol respectively. Most of fatty acids bound to human and bovine DBP are monounsaturated and saturated, mainly oleic and palmitic acids, which together account for 50% of the total of fatty acids in both species. By contrast, polyunsaturated fatty acids represented a minor component, less than 5%.     

Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

Interaction of rat alpha-fetoprotein and albumin with polyunsaturated and other fatty acids: determination of apparent association constants

The interaction of fatty acids with rat alpha-fetoprotein and albumin was measured using a partition equilibrium method. alpha-Fetoprotein (AFP) displays one high-affinity binding site for fatty acids and albumin near two binding sites. The AFP association constants for most fatty acids were similar to those of albumin (in the 10(7) M-1 range) whereas for docosahexaenoic acid it was 9.7 x 10(8) M-1, about 50-fold higher than that corresponding to albumin. This difference justifies docosahexaenoic acid in fetal or neonatal serum being mainly bound to AFP and can indicate a highly specific role of AFP in the transport of this fatty acid.