Structural organization of the twin-arginine translocation system in Streptomyces lividans

The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.     

Effect of different zinc sources and levels on inhibition of the apoptosis induced by glucocorticoid of thymocytes in vitro

This experiment was conducted to investigate the effects of zinc sulfate and zinc methionine (Zn-Met) and their levels on apoptosis induced by glucocorticoid of thymocytes and the possible mechanism. Dexamethasone was used to make the apoptosis model of thymocytes; zinc sulfate and zinc methionine were supplemented to the medium at levels of 0,50, 100, 500, and 1000 μM. The activity of cells,Cu,Zn superoxide dismutase (Cu,Zn-SOD), DNA ladder pattern, intracellular calcium concentration, and the percentage of apoptosis nuclei were determined. Both ZnSO₄ and Zn-Met could modulate apoptosis; they inhibited apoptosis and decreased DNA fragmentation. The regulation was concentration dependent. At levels of 50 and 100 μM, the effect of Zn-Met on inhibiting apoptosis was less efficient than that of ZnSO₄ (p<0.05), but the activity of the cells cultured with Zn-Met was higher than those cultured with ZnSO₄; they showed no difference in modulating apoptosis when added at levels of 500 and 1000 μM to the medium (p>0.05). Intracellular calcium concentrations of cells cultured with Zn-Met were higher than those cultured with ZnSO₄ at the same levels. Zinc supplementation decreased the concentration of intracellular calcium significantly (p<0.05) and increased the activity of Cu,Zn-SOD in the extract of the cells (p<0.05). Both zinc sulfate and Zn-Met could modulate apoptosis of thymocytes induced by glucocorticoid; the mechanism might involve the exchange of intracellular calcium, the redox of cells, and the two forms of zinc might go different ways in the regulations.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

脑啡肽增强胶质细胞的神经营养作用与NO生成减少有关

本文在ＳＤ大鼠大脑皮层胶质细胞神经元共培养模式上,以神经元存活、突起生长、生长相关蛋白４３（ｇｒｏｗｔｈａｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ４３,ＧＡＰ４３）ｍＲＮＡ的表达为指标,观察了脑啡肽对胶质细胞神经营养作用的影响,并对其机理作了初步探讨。结果表明,经脑啡肽处理的胶质细胞能使神经元的存活计数增加２８％（Ｐ＜００５）,单个神经元突起总长度增加１１％（Ｐ＜００５）,最长突起长度增加１６％（Ｐ＜００５）,ＧＡＰ４３ｍＲＮＡ的表达增加２６％（Ｐ＜００５）。然后又观察了脑啡肽（１０－６～１０－１２ｍｏｌ／Ｌ）对培养胶质细胞生成一氧化氮（ＮＯ）的影响。结果表明,浓度为１０－８,１０－１０ｍｏｌ／Ｌ的脑啡肽能明显抑制其生成（Ｐ＜００５）。结果提示,脑啡肽可能增强胶质细胞的神经营养作用,其机制之一可能是通过抑制胶质细胞ＮＯ的生成。     

Pyrophosphate and Adenosine 5′-Diphosphate Synthesis from Phospho(enol)pyruvate: Catalysis by Phosphate Minerals and Modulation by Dimethyl Sulfoxide

Phospho(enol)pyruvate (PEP) undergoes transphosphorylation to form pyrophosphate (PPi) and adenosine 5′-diphosphate (5′-ADP) with high yields in the presence of an adsorbent surface of calcium phosphate (Pi.Ca), which is considered to be an ancient mineral with catalytic properties. PPi formation is a result of the phosphorolytic cleavage of the enol phosphate group of PEP by precipitated Pi. The synthesis of PPi is dependent on the amount of the solid matrix; it increases with the amount of adsorbed PEP and upon addition of dimethyl sulfoxide (Me₂SO), a molecule with high dipolar moment. Although it is saturated with PEP at neutral pH, the phosphorylating Pi.Ca surface becomes effective only in alkaline conditions. In a parallel reaction, PEP phosphorylates 5′-AMP to 5′-ADP with a yield that is sevenfold higher in the presence of the Pi.Ca surface than in its absence, indicating that the solid matrix promotes interaction between adsorbed molecules with a high potential for phosphoryl transfer. In contrast to phosphorolysis, this latter reaction is stimulated by Me₂SO only in homogeneous solution. It is concluded that phosphate minerals may have coadjuvated in reactions involving different phosphorylated compounds and that molecules with high dipolar moment may have acted in mildly alkaline, primitive aqueous environments to modulate phosphoryl transfer reactions catalyzed by phosphate minerals. Received: 31 January 1996 / Revised: 31 May 1996     

Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro

Summary The growth of bovine aortic smooth muscle and endothelial cells was studied after exposure to dimethyl sulfoxide (DMSO) or its major metabolite, dimethyl sulfone (DMSO₂). Both compounds caused a dose-dependent inhibition of cell growth as determined by [³H]thymidine incorporation and by counting the number of cells with time of exposure in culture. The IC₅₀ of DMSO (concentration which produces 50% inhibition of growth) was 1% for smooth muscle cells and 2.9% for endothelial cells. Similarly, the IC₅₀ of DMSO₂ was also 1% for smooth muscle cells, but was 1.8% for endothelial cells. After a 4-d exposure to either compound, the growth inhibition of smooth muscle cells was completely reversible at 1%, partially reversible at 2 to 3% and completely irreversible at 4%. By comparison, inhibition of endothelial cell growth was completely reversible up to 4% of either compound. It is concluded that the growth of smooth muscle cells was similarly inhibited by DMSO, and DMSO₂, but that smooth muscle cells were more susceptible than endothelial cells to the growth inhibitory effects of these compounds. In addition, DMSO₂ was a more potent inhibitor of cell growth than DMSO and its growth inhibition was less reversible than that produced by DMSO.     

Plasma Lipid Peroxides in Murine Sepsis —Sex Differences and Effect of Antioxidative/Anti-Inflammatory Therapy

In order to investigate the influence of antioxidative anti-inflammatory combination therapy (AACT) with dimethyl sulfoxide (DMSO). chlorpromaittic (CPZ) and vitamin E upon the activity of the inflammation. plasma lipid peroxide was measured as thiobarbituric acid reactive substance (TBARS) 12hrs postoperatively in the moclitied cecal ligation sepsis model in the mouse.

Significantly higher TBARS levels were found in the male control group (13.7 ± 0.7nmol MDA/ml) than in the female control group (11.6 ± 0.6nmol MDA/ml).

The operated male group had significantly higher TBARS levels (16.2 ± 0.6 nmol MDA/ml) than the unoperdted male control group (13.7 ± 0.7nmol MDA/ml). No increase of TBARS levels was observed in the operated female group.

Both male and female operated group. when postoperatively treated with AACT had the same TBARS level as the not operated male or female control group.

Survival curves of operated male and female group did not demonstrate any significant difference. The survival was better in an operated male and an operated female group. when postoperatively treated with AACT.

It was concluded that the applied TBARS test IS too insensitive to follow the activity of the inflammation and has no predictive value for the outcome of sepsis in this model.     

Different selectivities of oxidants during oxidation of methionine residues in the α-1-proteinase inhibitor

Oxidation of the reactive site methionine (Met) in α-1-proteinase inhibitor (α-1-PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α-1-PI, we measured the molar ratio Met(O)/α-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H₂O₂/chloride system and the related compound NH₂Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol α-1-PI during inactivation. These oxidants attack preferentially one Met residue in α-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30–50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of α-1-PI activity in vivo.     

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.