The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain

Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.     

Truncation of peptide deformylase reduces the growth rate and stabilizes solvent production in Clostridium beijerinckii NCIMB 8052

The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn1545 mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the fms (def) gene, which encodes peptide deformylase (PDF). The C. beijerinckii fms gene product contains four diagnostic residues involved in the Zn2+ coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from Escherichia coli and Thermus thermophilus, the C. beijerinckii PDF can apparently tolerate N-terminal truncation. The Tn1545 insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retains at least partial function, and it complements an fms(Ts) strain of E. coli. Northern hybridizations indicate that the mutant gene is actively transcribed in C. beijerinckii. This can only occur from a previously unsuspected, outwardly directed promoter located close to the right end of Tn1545. The Tn1545 insertion in fms causes a reduction in the growth rate of C. beijerinckii, and, associated with this, the bacteria display an enhanced stability of solvent production. The latter phenotype can be mimicked in the wild type by reducing the growth rate. Therefore, the observed amelioration of degeneration in the mutants is probably due to their reduced growth rates.     

Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration

OBJECTIVES: The purpose of this study was to test the hypothesis that long-term supplementation with Vitamin E improves endothelium-dependent relaxation in hypercholesterolemia patients and/or chronic smoking, two risk factors that have been shown to be associated with increased radical formation. BACKGROUND: Experimental evidence suggests that oxidized low density lipoprotein (LDL) impairs endothelium-dependent relaxation, and vitamin E, a lipid-soluble antioxidant, reduces the oxidation of LDL. METHODS: Thirteen subjects with hypercholesterolemia, 14 smokers and 15 hypercholesterolemic smokers were enrolled in a double-blind, placebo-controlled study. After baseline measurements of plasma autoantibodies against oxidized LDL and assessment of endothelium-dependent relaxation using intra-arterial forearm infusions of acetylcholine, participants within each group were randomly assigned in a 1:2 fashion to receive either placebo or vitamin E for 4 months, when plasma levels of autoantibodies against oxidized LDL and vascular function were reassessed. RESULTS: Vitamin E significantly augmented endothelium-dependent relaxation in hypercholesterolemic smokers but not in patients with either hypercholesterolemia or chronic smoking. At baseline, hypercholesterolemic smokers had significantly higher autoantibody levels against oxidized LDL (compared with the other two groups), which were significantly reduced after 4 months of vitamin E supplementation. There was a significant relationship between improvement in acetylcholine-induced vasodilation and the change in autoantibody titer against oxidized LDL (r = -0.59; p = 0.002). CONCLUSIONS: Long-term vitamin E supplementation improves endothelium-dependent relaxation in forearm resistance vessels of hypercholesterolemic smokers, which are characterized by increased levels of autoantibodies against oxidized LDL. These findings may suggest that the beneficial effect of vitamin E is confined to subjects with increased exposure to oxidized LDL.     

Evidence for an heterogeneous glycosylation of the Clostridium tyrobutyricum ATCC 25755 flagellin

Glycosylation analysis of the flagellin from the Gram-positive species Clostridium tyrobutyricum has been supplemented. Amino acid analysis of the glycopeptides obtained after pronase digestion of flagellin indicated that O-glycosylation which was previously demonstrated after nonreductive beta-elimination, probably occurred via the hydroxyl group of serine. Otherwise, beta-elimination partly deglycosylated flagellin. After this treatment carbohydrates were still linked to protein as shown by a digoxigenin-hydrazide labelling. Therefore, in addition to linkages via serine, alkaline resistant linkages exist on the flagellin and some glycans may be linked to the protein core via the amide nitrogen of asparagine or via the hydroxyl group of tyrosine. Furthermore, according to an immunological analysis, glycans attached to flagellin via alkaline sensitive linkages may be different from those attached via alkaline resistant linkages.     

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 microF, and 600 omega. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10(1) transformants/microgram DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained.     

Isolation and characterization of a mutant of Pseudomonas aureofaciens ATCC 15926 with an increased capacity for synthesis of pyrrolnitrin

A mutant having a 30-fold increased ability to synthesize pyrrolnitrin was isolated from Pseudomonas aureofaciens ATCC 15926 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant strain also differed from the parent strain in pigmentation and in its inability to catabolize anthranilic acid and the benzene moiety of tryptophan and kynurenine.     

Leaching of lead cakes of zinc production in a complexing solvent

The process of leaching of lead cakes of zinc production in a complexing solvent—trilon B, which makes it possible to selectively extract lead into the solution—is studied with the use of mathematical modeling of the experiment. Its optimum conditions—pH, liquid-to-solid ratio within pulp, and temperature—are determined. The developed flow chart of cake processing provides zinc removal as a single product, recycling of zinc to the basic production, and reduction of current expenditure due to solvent regeneration.     

Transforming growth factor beta 1 induces squamous carcinoma cell variants with increased metastatic abilities and a disorganized cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial-fibroblastic conversion when cultured in the presence of TGF-beta1. This conversion is associated in vivo with a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-beta1. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell-cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell-cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.     

Effect of isoleucine on toxin production by Clostridium difficile in a defined medium

Supplementation of a carbohydrate-free minimal medium with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production by Clostridium difficile VPI 10463. The effect of isoleucine was further examined. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. Amino acid analysis of the culture revealed that, at 100 mM isoleucine, consumption of isoleucine was remarkably increased. During the incubation period when toxin titers increased markedly but bacterial growth was declining, isoleucine, leucine and cysteine were taken up preferentially and alanine and cystathionine, which were not found at 1 mM isoleucine, were produced in large quantities. These findings suggest that isoleucine may play an important role in toxin production by C. difficile and that alanine and cystathionine production may be co-regulated with the toxin production in the absence of fermentable carbohydrates.     

Nickel-free steel of increased strength and corrosion resistance for freight-car production

Nickel-free 10ΓAΦДΠ1 steel of elevated strength, low-temperature strength, and low-temperature resistance has been developed. On the basis of the new steel, a production technology for rolled sheet and cold-bent profiles (thickness 3.6–5.0 mm) for freight-car production is proposed. In the electrosmelting shop at OAO Magnitogorskii Metallurgicheskii Kombinat (MMK), a batch of the new steel (160 t) has been produced and rolled to 4.5 × 1500 mm sheet in the 2500 mill. Coils of this steel are used to produce bent profiles for freight-car production. The new 10ΓAΦДΠ steel surpasses mass-produced 10XHДΠ 1 steel in terms of its suitability for freight-car components.     

Decreased flow-induced dilation and increased production of cGMP in spontaneously hypertensive rats

-We investigated flow (shear stress)- and agonist-induced cGMP release in mesenteric vascular beds of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The mesenteric vascular bed was perfused in situ with Tyrode's solution. Vascular relaxation and cGMP release in the perfusate were determined on stimulation by flow or by acetylcholine (0.1 micromol/L) or sodium nitroprusside (0.1 mmol/L). Flow-induced release of cGMP was significantly greater in SHR than in WKY (P<0.01), despite a lower flow-induced dilation in SHR. In both strains, NG-nitro-L-arginine methyl ester (L-NAME) completely inhibited cGMP release in response to flow (P<0.001), although flow-induced dilation was not affected by L-NAME in SHR. Moreover, the activity of the constitutive nitric oxide synthase (NOS) was significantly greater in SHR (82+/-3.5 fmol/min) than in WKY (66+/-3.5 fmol/min; P<0.05) and was associated with increased expression of endothelial NOS mRNA in SHR. Sodium nitroprusside induced larger increases in cGMP release in SHR (3593+/-304 fmol/min) than in WKY (2467+/-302 fmol/min; P<0.05). The release of cGMP in response to acetylcholine was significantly lower in SHR (292+/-80 fmol/min) than in WKY (798+/-218 fmol/min; P<0.05) in parallel with smaller acetylcholine-induced relaxation in SHR. Despite increased cGMP production in response to flow and NOS activity, flow-induced dilation was decreased in SHR, suggesting an upregulation of the NO/cGMP pathway to compensate for the increased vascular tone in SHR.     

Dissipation of the calcium gradient in human erythrocytes results in increased heat production

Heat production rates were measured by a microcalorimetric method in suspended human erythrocytes in the absence and presence of different concentrations of the divalent cationophore A23187. Determinations were carried out during 60 min under static conditions on erythrocytes incubated in various isotonic media at 37 degrees C, pH 7.35. In incubations containing the ionophore, time-power curves showed an early peak followed by a descending slope levelling off at a steady state after 30-60 min. In contrast, the controls lacked the early peak, showing hyperbolic ascending curve profiles before reaching steady state. The appearance of the early peak in the presence of ionophore was dependent upon the composition of the medium, both Mg2+ ions and glucose being decisive. Likewise, dose-response relationships concerning heat production at 60 min depended on the composition of the media. In a basic incubation medium lacking Mg2+ and glucose, no effect was seen on heat production by the ionophore (1-3 mumol/l). Rather modest effects were obtained by the ionophore at 2 and 3 mumol/l when Mg2+ was present. A clear-cut dose-response relationship was observed in a Mg2+ and glucose enriched medium for the ionophore from 1-3 mumol/l. The significant increase in heat production observed at 60 min with 2 mumol/l of A23187 in the Mg2+ and glucose enriched medium was abolished by 1 mmol/l EGTA. Calmidazolium, a calmodulin antagonist, could only marginally reduce the ionophoric effect on heat production. It was concluded that the appearance of the early peak was not the result of an increase in glycolytic rate but rather a consequence of the ionophoric action on the Ca2+ gradient.     

Altered production of laminin and nidogen by high and low metastatic variants of murine melanoma cells

The launching of cheap, pocket-sized spirometers, with data storage capability, has made patient-administered sequential spirometry (PASS) an attractive method of monitoring ventilatory capacity. At present, little information is available on the quality of PASS, compared to laboratory spirometry. The aim of this study was to investigate whether patients could perform PASS without loss of reliability and reproducibility as compared with traditional laboratory spirometry. Ten healthy volunteers performed spirometry for 1 month and 10 emphysematous patients with alpha 1-antitrypsin deficiency (type PiZ) performed spirometry twice daily for up to 2 yr. To fulfil Good Clinical Practice criteria on full data documentation, a traditional direct recording spirometer, the Vitalograph R-model, was used. A decompression device was used for calibration and a 3.8% annual drift in volume registration was noted. This drift was largest for the first year. After training, all patients were able to perform unsupervised spirometry, producing technically correct forced expiratory curves. Reproducibility of FEV1 and FVC obtained by PASS was found to be as good as for laboratory spirometry. After adjustment for the diurnal variation, the residual variation of FEV1 was 2.5% (range 1.6-4.2%) for healthy volunteers and 5.6% (range 4.2-7.7%) for emphysematous patients. Forced vital capacity showed the same pattern. In conclusion, PASS is possible in highly motivated individuals without loss of reliability and reproducibility when compared to laboratory spirometry.     

CD4 T lymphocytes from patients with chronic fatigue syndrome have decreased interferon-gamma production and increased sensitivity to dexamethasone

A disturbed hypothalamus-pituitary-adrenal gland axis and alterations at the immune system level have been observed in patients with chronic fatigue syndrome (CFS). Glucocorticoids are known to modulate T cell responses; therefore, purified CD4 T cells from CFS patients were studied to determine whether they have an altered sensitivity to dexamethasone (DEX). CD4 T cells from CFS patients produced less interferon-gamma than did cells from controls; by contrast, interleukin-4 production and cell proliferation were comparable. With CD4 T cells from CFS patients (compared with cells from controls), a 10- to 20-fold lower DEX concentration was needed to achieve 50% inhibition of interleukin-4 production and proliferation, indicating an increased sensitivity to DEX in CFS patients. Surprisingly, interferon-gamma production in patients and controls was equally sensitive to DEX. A differential sensitivity of cytokines or CD4 T cell subsets to glucocorticoids might explain an altered immunologic function in CFS patients.     

Correlation between production of listeriolysin O by variants of Listeria monocytogenes and their virulence for rabbits

L. monocytogenes serovar 1/2a NCTC 7973 was passaged through rabbits and the severity of infection at each passage was determined by counting viable bacteria from infested organs and recording the time of death. A comparative evaluation of the levels of hemolysin produced in vitro by the original and six variant cultures (V1-V6) was done by determination of hemolytic units (CHU). While virulence of the cultures enhanced at each passage (2.2 x 10(9) CFU/g of the spleen for V6 as compared to 5.0 x 10(6) CFU/g spleen for the parent culture), the CHU decreased considerably, 3 CHU for the V6 as compared to 40 CHU for the parent strain. The results suggest that the level of in vitro production of listeriolysin may not parallel the pathogenicity of L. monocytogenes for rabbits.     

溶剂萃取ICP-AES法测定钢中磷

由于磷在可见光范围内的分析线受其他元素干扰情况严重 ,用ＩＣＰ -ＡＥＳ直接准确测定很困难。用驱气充氩系统测定磷线 1 78 2 2 4ｎｍ的峰值强度 ,可获得准确结果。但充气需要时间长 ,耗气量大 ,样品量少时很不经济。本文研究了一种不需充氩的方法。在 0 6 5～ 1 6 3ｍｏｌ/Ｌ硝酸介质中 ,磷与钼酸铵生成的磷钼杂多酸被乙酸丁酯萃取 ,而其他杂多酸盐和过量的钼酸铵不会被萃取。用强还原剂二氯化锡可将磷钼杂多酸还原为磷钼杂多蓝 ,并将其反萃取到水相[1 ] ,以钼为测定元素[2 ] ,用ＩＣＰ -ＡＥＳ法测定水相磷钼酸中钼 ,间接测定了磷的含…