Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

Production of 5α- and 5β-dihydrotestosterone by isolated human axillary bacteria

Abstract The metabolism of testosterone by coryneform bacteria in vitro has been studied. Metabolites identified after derivatization by capillary gas chromatography and further by combined gas chromatography-mass spectrometry were 17β-hydroxy-5α-androstan-3-one and 17β-hydroxy-5β-androstan-3-one. The mass spectral characteristics of the methyl oxime trimethylsilyl ethers of all the 17-hydroxy-androstan-3-one and 3-hydroxy-androstan-17-one isomers are recorded.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Regulation of δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the α-aminoadipate pool size

The effect of changes in the intracellular concentration of alpha-aminoadipate on the formation of alpha-aminoadipyl-cysteinyl-valine (ACV) and isopenicillin N (IPN)--two intermediates of penicillin biosynthesis--by strains of Penicillium chrysogenum has been investigated by measuring the incorporation of radioactivity from (6-14C)-alpha-aminoadipate into cellular 14C-ACV and 14C-IPN. No ACV or IPN were found in any strain during cultivation on glucose, but were clearly detected in all three strains during growth on lactose, displaying increased formation in strains exhibiting increased penicillin productivity and increased intracellular alpha-aminoadipate pools. ACV and IPN formation was affected by subjected P. chrysogenum mycelia to either general amino acid control (by addition of amitrol) or by exogenous addition of 5 mM L-lysine. In all cases, the changes observed paralleled the changes in the intracellular alpha-aminoadipate pool. These results are consistent with the alpha-aminoadipate pool limiting the biosynthesis of ACV and IPN and hence penicillin biosynthesis in the present strains of P. chrysogenum.     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Extent of humification of anthracene, fluoranthene, and benzo[α]pyrene by Pleurotus ostreatus during growth in PAH-contaminated soils

The white-rot fungus Pleurotus ostreatus catalysed some humification of anthracene, benzo[ a ]pyrene and fluoranthene in two polycyclic aromatic hydrocarbon (PAH)-contaminated soils, one from a former manufactured gas facility and one from an abandoned electric coking plant. However, the extent of humification of PAH observed in these experiments was considerably less than that previously reported for other pollutants, such as chlorophenols. Addition of surfactants and related amendments significantly enhanced PAH removal from both soils by P. ostreatus , although humification of PAH was not always enhanced under these conditions.     

Long-chain α-hydroxy-(ω-1)-oxo fatty acids and α-hydroxy-1,ω-dioic fatty acids are cell wall constituents of Legionella (L. jordanis, L. maceachernii and L. micdadei)

Abstract Four long-chain fatty acids, 2-hydroxy-27-oxo-octacosanoic acid ( n 28:0(2-OH,27-oxo)), 2-hydroxy-29-oxo-triacontanoic acid ( n 30:0(2-OH,29-oxo)), 2-hydroxy-heptacosane-1,27-dioic acid (27:0(2-OH)-dioic) and 2-hydroxy-nonacosane-1,29-dioic acid (29:0(2-OH)-dioic) were identified by GLC-MS analysis in the phenol-chloroform-petroleum ether (PCP) extracts of Legionella jordanis, L. maceachernii and L. micdadei indicating that they are constituents of lipopolysaccharide. Moreover, five long-chain fatty acids (28:0(27-OH), 28:0(27-oxo), 30:0(29-oxo), 27:0-dioic and 29:0-dioic) previously identified in L. pneumophila (Moll, H. et al., FEMS Microbiol. Lett., 97 (1992), 1–6) were also found in these species. This is to our knowledge the first report on the existence of long chain 2-hydroxylated (ω-1)-oxo fatty acids and 2-hydroxylated 1,ω-dioic fatty acids.     

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α-N-Acetyl Derivative

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH₂ terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ?15 fmol of β-endorphin per incubation tube, i.e., ? 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH₂ terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.     

Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate, and of the glucuronides of testosterone and 17β-oestradiol, by urine of reproductively mature male and female rainbow trout (Oncorhynchus myklss)

Levels of sulphated 17α20β-dihydroxy-4-pregnen-3-one (17,20 β -P; the oocyte maturation inducing steroid) in blood plasmas of sexually mature male and female rainbow trout Oncorhynchus mykiss , were very low in comparison to those of the free steroid. However, relatively large amounts were found in urine of both sexes.
Catheters were inserted into the urinary bladders of unovulated and ovulated females and of ripe-running males, and the fish then placed in spawning channels. Three-hourly urine samples were collected between 09.00 and 18.00 hours and then a 15-h sample between 18.00 and 09.00 hours the next morning. Measurements were made of 17,20 β -P-sulphale, testosterone glucuronide (T-G) and 17 β -oestradiol glucuronide (E2-G). In females, the highest rates of excretion of E2-G, T-G and 17,20 β -P-sulphate were found in unovulated, ovulating and ovulated females, respectively. The rates of excretion of 17,20 β -P-sulphate, T-G and E2-G in ovulated females were unaffected by the presence of a male. id males, however, there was a sharp increase in the rate of excretion of 17,20 β -P-suiphate and T-G in fish which were paired with an ovutated (nesting) female. A similar increase was found in males injected with male trout pituitary extract.     

S100a0 (αα) Protein, a Calcium-Binding Protein, Is Localized in the Slow-Twitch Muscle Fiber

We previously showed that, in contrast to the distribution of S100b (beta beta), S100a0 (alpha alpha) is mainly present in human skeletal and heart muscles at the level of 1-2 micrograms/mg of soluble protein and is universally distributed at high levels in skeletal and heart muscles of various mammals. To elucidate cellular and ultrastructural localizations of the alpha subunit of S100 protein (S100-alpha) in skeletal muscle, we used immunohistochemical and enzyme immunoassay methods. The immunohistochemical study revealed that S100-alpha is mainly localized in slow-twitch muscle fibers, whereas the beta subunit of S100 protein (S100-beta) was not detected in both types of muscle fibers, an observation indicating that the predominant form of S100 protein in the slow-twitch muscle fiber is not S100a or S100b, but S100a0. The quantitative analysis using enzyme immunoassay corroborates the immunohistochemical finding: The S100-alpha concentration of mouse soleus muscle (mainly composed of slow-twitch muscle fibers) is about threefold higher than that of mouse rectus femoris muscle (mainly composed of fast-twitch muscle fibers). At the ultrastructural level, S100-alpha is associated with polysomes, sarcoplasmic reticulum, the plasma membrane, the pellicle around lipid droplets, the outer membrane of mitochondria, and thin and thick filaments, by immunoelectron microscopy.     

Production of aromatic α-hydroxyacids by epimastigotes of Trypanosoma cruzi, and its possible role in NADH reoxidation

Abstract Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p -hydrocyphenyllactic acids, presumbly arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic α-hydroxyacid dehydrogenase.     

Changes in α-Tubulin and Actin Gene Expression During Optic Nerve Regeneration in Frog Retina

The optic nerve of the bullfrog was transected and the regeneration process was investigated. We previously reported that alpha-tubulin mRNA in the retina increased to a maximum 1-2 h after optic nerve transection with no specific change in actin mRNA. In the present investigation, we examined the long-term effect of optic nerve transection. Northern blot analysis revealed that alpha-tubulin mRNA increased again gradually after the rapid and transient increase and actin mRNA increased to a maximum at 7 days (more than twofold compared to the control retinas). The period during which actin mRNA reaches a maximal increase almost corresponds to the time lag between the axotomy and the initiation of axonal outgrowth. The main cytoskeletons of neuronal growth cones have been shown to consist of actin-containing microfilaments. Therefore, the transient increase of actin mRNA may have a relationship to the initial outgrowth of axons. On the other hand, the rapid and transient increase of alpha-tubulin mRNA observed in our previous studies is probably one of the initial responses of retinal ganglion cells to the axotomy, and the gradual increase in alpha-tubulin mRNA observed in this study can probably be interpreted as provision of the structural materials necessary for axonal elongation.     

用EROD研究苯并芘和六氯苯的联合毒性作用

用7-乙氧基异叻唑酮-脱乙基酶(EROD)检测的方法,研究了苯并芘和六氯苯对日本青鳉肝脏EROD酶的比活力的影响。结果表明,苯并芘和六氯苯对EROD酶的比活力均有激活作用,在实验浓度范围内,EROD酶的比活力与两者浓度之间存在剂量-效应关系。苯并芘和六氯苯表现为一定的协同作用。实验同时发现日本青鳉在六氯苯和苯并芘中暴露后,EROD酶的比活力开始有一个短暂的降低,然后持续升高。对六氯苯和苯并芘暴露的最佳时间进行了探讨。