β-Lactamases from Actinopolyspora halophila,an extremely halophilic actinomycete

Actinopolyspora halophila, an extremely halophilic actinomycete, produced both cellular and exocellular -lactamases when grown in liquid media containing 20% (w/v) NaCl. Maximal exocellular -lactamase production occurred at 48 h growth and exceeded cellular enzyme levels 7-fold. Maximal cellular -lactamase was observed at 72 h as cultures achieved full growth.Both -lactamases were purified to molecular homogeneity by a sequence involving gel filtration on Bio-Gel P-100, DEAE Bio-Gel A chromatography, preparative isoelectric focusing, and gel filtration on Sephadex G-75. Cellular -lactamase was purified 99-fold with 12% recovery and had a molecular weight of 42,200, and an isoelectric point of 4.15. Exocellular -lactamase was 12-fold purified with 1.2% recovery of initial activity and had a molecular weight of 38,000 and an isoelectric point of 3.85. Its specific activity was 7-fold greater than that of the cellular enzyme.A variety of penicillin and cephalosporin substrates were degraded by both enzymes. While the cellular -lactamase degraded phenoxymethylpenicillin, methicillin, and cephaloglycin most efficiently, the exocellular enzyme was most active against methicillin, carbenicillin, ampicillin, and cephalosporin C. Both enzymes were stimulated and protected from thermal deactivation by NaCl but not KCl or MgCl₂-Neither enzyme was inhibited by iodine.Abbreviations IEF Isoelectric focusing - SDS-PAGE sodium dodecylsulphate-polyacrylamide electrophoresis - PEG polyethyleneglycol - DTT dithiothreitol - DEAE diethylaminoethyl Issued as NRCC 25164     

Effects of glycine supplement on protein production and release in recombinant Escherichia coli

Summary The effect of glycine supplement to growth media on protein expression and release in a recombinant strain RR1 of E. coli was investigated. Addition of glycine to the growth media in moderate amount (up to 1%) was observed to enhance significantly the release of periplasmic proteins from the cell to the broth. The extracellular activities of the model enzymes -amylase and -lactamase were increased by a factor of 16.3 and 3.8 respectively in the presence of glycine. These activities corresponded to about 50% of the total production for each protein. Furthermore, with glycine supplement the total enzyme activity of both -amylase, -lactamase as well as -galactosidase were increased by a factor of about 2.5. Cell growth characteristics and low extracellular activity of the cytoplasmic protein -galactosidase are indicative that glycine does not cause significant cell-lysis for a concentration below 0.7%.     

Elimination of the disulphide bond alters the conformation of mature lipo-β-lactamase in yeast

Summary When expressed in Saccharomyces cerevisiae the precursor of the hybrid prokaryotic protein lipo--lactamase is accumulated in reduced form whereas the majority of the mature form contains an intra molecular disulphide bond (oxidized form). We have previously shown that mature mutant lipo--lactamases in which the cysteine residue 131 was changed to either tyrosine or threonine lack the capacity to form a disulphide bond but are nevertheless processed and secreted efficiently in yeast. Here we show that these mutant mature lipo--lactamases, in yeast cell extracts, exhibit a tenfold lower -lactamase-specific activity than the wild-type protein and that the mature mutant proteins are more susceptible to trypsin digestion. Thus, elimination of the disulphide bond alters the conformation of mature lipo--lactamase in yeast.Correspondence to: O. Pines     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Trimetazidine increases phosppholipid turnover in ventricular myocyte

Ehrlich ascites tumor cells incorporate [methyl-3H]thymidine into DNA independently of exogenous growth factors or fetal calf serum. Using an acid/ethanol extraction procedure we have obtained from these tumor cells a fraction that induces both the proliferation and the formation of cell foci by Swiss 3T3 mouse fibroblasts in the presence of insulin; inhibits the proliferation of Mv1Lu mink lung epithelial cells; and stimulates the growth of NRK rat kidney fibroblasts in soft-agar in the presence of epidermal growth factor. An antibody against transforming growth factor- (TGF) prevents both the tumor extract-induced proliferation of Swiss 3T3 fibroblasts and the tumor extract-induced proliferative arrest of Mv1Lu cells. The tumor cells secrete a TGF-like activity to the extracellular medium in a partially-activated form. However, authentic TGF does not affect their proliferation, and no TGF receptors were detected using [125I]TGF as a ligand. Therefore, the absence of TGF receptors with ligand-binding capacity in these tumor cells may bypass the negative control that this factor exerts on the proliferation of their normal cell counterparts.     

Decreases in Plasma Aβ1-40 Levels with Aging in Non-Demented Elderly with ApoE-ε4 Allele

This report examines plasma amyloid proteins A40 and A42 and apolipoprotein E (apoE) levels and their relationships with age in non-demented older adults with (N = 32) or without the apoE-4 allele (N = 94). A levels did not differ between the groups whereas the 4 allele was associated with a significant reduction in plasma apoE. In subjects with the 4 allele, increasing age was associated with significant reduction in plasma A40. Subjects without the 4 allele showed a significant positive correlation between A40 and A42 levels. There was also a significant correlation between plasma A40 and apoE levels in all subjects.     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Factors affecting the synthesis and distribution ofβ-lactamase inMycobacterium smegmatis SN2 cultures

A high level of extracellular -lactamase activity was detected in cultures ofMycobacterium smegmatis SN2. The extracellular distribution of the enzyme varied with growth conditions such as additional carbon source and pH of the medium. Addition of chloramphenicol tothe culture inhibited the increase in the extracellular -lactamase activity. Cell wall damage or autolysis may be responsible for the extracellular -lactamase activity.     

Degradation of 5-pregnene-3β, 20β-diol by a Nocardia sp.

Summary With growing cells of a Nocardia sp., isolated from soil, the degradation of 5-pregnene-3, 20-diol into 3-[5-oxo-7a-methyl-1 (1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid was investigated. The results show that iron is essential for production of the perhydroindanpropionic acid, that this production is greatly enhanced by the presence of calcium and that it is maximal in the pH range 7.0–7.5.Abbreviations used in the text PD 5-pregnene-3, 20-diol (pregnendiol) - PDSA 3-[5-oxo-7a-methyl-1(1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid (pregnendiol-secoacid) - PSA 3-[5-oxo-7a-methyl-1-acetyl-3a-perhydroindane-4]-propionic acid (progesterone-secoacid) - EDTA Ethylendiamintetracetic acid - DMSO Dimethylsulfoxide     

Alzheimer’s Disease: Soluble Oligomeric Aβ(1–40) Peptide in Membrane Mimic Environment from Solution NMR and Circular Dichroism Studies

Amyloid beta peptide (A) is a small peptide present in normal cells and aggregated A is the main constituent of the extracellular amyloid plaques found in Alzheimers disease (AD) brain. Recent studies suggest that soluble A oligomers are neurotoxic rather than amyloid fibrils found in amyloid plaques. This study using multidimensional NMR spectroscopy and circular dichroism (CD) provides the first direct evidence that alterations in membrane structure can trigger the conversion of soluble -helical monomeric A into oligomeric A in a -sheet conformation.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Extracellular β-lactamase from Escherichia coli

Summary Escherichia coli strain O 127: K63: (B8): H—was grown in nutrient broth (Difco). Penicillinase activity was found in the culture supernatant after only five hours of incubation, i.e. during the exponential phase of growth. At this phase the levels of typical intracellular markers, did not indicate cell lysis or gross cell damage. The bioautographic revelation of penicillin-splitting enzymes on electropherograms of cell-free liquids confirmed the presence of one basic broad-spectrum -lactamase. This possibly extracellular -lactamase seems to be also present in the cellular extracts where it coexists with several other cell-bound penicillinases.     

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons

Summary Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN) and immune (IFN) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN than to IFN and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN plus IFN was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN and IFN differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN plus IFN can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN or IFN, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.     

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4–12-fold) stimulation of the activity of membrane-associated -glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of -glucan synthesis. Those products whose synthesis is stimulated are mainly -1,3-glucan, but there is also a considerable increase in -1,4-glucan. No -1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on -glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.Abbreviations UDP-glucose uridine-5-diphosphoglucose - PEG polyethylene glycol - BTP bistrispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane) - MES 2(N-morpholino)ethanesulfonic acid - VAL valinomycin     

Studies of two strains of Actinopolyspora halophila,an extremely halophilic actinomycete

Actinopolyspora halophila is an extremely halophilic actinomycete requiring a minimum salt concentration of 12% (w/v) for growth in liquid media at 37°C. During antibiotic sensitivity testing of the organism, an erythromycin-resistant phenotypic variant designated A. halophila ER, was isolated. This strain was approximately 60-fold more resistant to erythromycin than the wild-type strain, and, moreover, was capable of growth in 6% sodium chloride. Optimal growth of both strains in liquid media was observed in 20% salt.After resuspension in water for up to 20 days, the organism exhibited good growth in fresh salt-containing media. Cultures resuspended in salt-containing media, however, were subject to extensive cellular autolysis.In addition to resistance to -lactam antibiotics which was mediated by cellular and exocellular -lactamases, the organism was insensitive to all other tested antibiotics except amakacin, chloramphenicol, kanamycin, gentamycin, naladixic acid, streptomycin, and tetracycline. No plasmids were detected in either strain. A. halophila possessed high cellular levels of catalase, -lactamase, and -glucosidase. A variety of exocellular enzymes including protease, -lactamase, xylanase, and carboxymethyl cellulase, were secreted maximally into growth media containing 15% NaCl.Issued as NRCC 25148     

Degradation of β-lactam antibiotics by polyacrylamide-entrapped β-lactamase-producingE. coli cells

Summary Cells of a -lactamase producingE. coli strain were immobilized with acrylamide and lyophilized. The gel particles containing the entrapped cells were used like an immobilized enzyme to study the inactivation of -lactam antibiotics. The substrate profile of the -lactamase was determined and the action of -lactamase inhibitors studied.     

Analysis of the 5′ flanking sequence of the Gγ globin gene by denaturing gradient gel electrophoresis confirms the heterogeneity of the Bantu βs haplotype

Summary The GCTT polymorphism recently described at positions — 1106 and — 1105 in the 5 flanking region of the G globin gene for the Bantu ^S haplotype was analysed by denaturing gradient gel electrophoresis. We studied 108 ^S chromosome and 122 ^A chromosomes. The TT sequence was found as follows: in all of 80 chromosomes bearing the Bantu ^S haplotype with the 6-bp deletion -400 nt from the G gene in 3 out of 5 Bantu ^S chromosomes without the deletion, in 1 out of 122 ^A chromosomes from different ethnic origins but in none of 23 ^S chromosomes bearing the Senegal, Benin or Cameroon haplotypes. These results confirm the heterogeneity of the Bantu ^S haplotype and allow a tentative evolutionary sequence for the different alleles at this locus to be presented.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.