Effects of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene

Primary culture of vascular smooth muscle cells (VSMC) from rat aorta was used for the study of the effect of different peptides derived from proadrenomedullin on the expression of adrenomedullin (ADM) gene. ADM and preproADM(22-41) (PAMP) secreted by VSMC were measured by radioimmunoassay, and ADM mRNA in VSMC was determined by quantitative RT-PCR. After the incubation of VSMC in 10(-7)M ADM for 24h, PAMP in the medium and ADM mRNA in the VSMC were decreased by 34 and 41.3%, respectively, and cAMP concentration in the VSMC was increased by 385%. After the incubation of VSMC in 10(-7)M PAMP for 24h, ADM in the medium and ADM mRNA in the VSMC were decreased by 12.2 and 39.1%, respectively, and cAMP concentration in the VSMC was increased by 67%. The decreased ADM mRNA in VSMC induced by the ADM and PAMP treatment was completely reversed by the pre-treatment of the cells in 10(-7)M protein kinase inhibitor for 30 min. After the incubation of VSMC in 10(-7)M preproADM(153-185) (ADT) for 24h, however, ADM in the medium and ADM mRNA in the VSMC were increased by 21 and 35.2%. The increased ADM mRNA in VSMC induced by the ADT treatment was partially blocked by the co-incubation in ADM and ADT, and was totally blocked by the co-incubation in PAMP+ADM and ADT, but was not blocked by the co-incubation in PAMP and ADT. Our results suggest that the four peptides derived from proadrenomedullin may have different effects, possibly through a cAMP-dependent pathway, on the expression of ADM gene.     

Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

植物生长调节物质IP－1号对木薯产量及其生物性状的影响

１９９０和１９９１年在木薯（ＭａｎｉｈｏｔｅｓｃｕｌｅｎｔａＣｒａｎｔｚ）生长期以植物生长调节物质ＩＰ－１号０,２０,３０和４０ｐｐｍ进行叶面喷洒,结果表明：３０ｐｐｍ处理可使木薯块根产量平均增加５４．４４％,块根淀粉含量平均提高２０．８１％。单株最大薯重提高３１．５５％,块根数增加２１．１７％,块根长度增长１７．６２％,地上部鲜重增加３４．３６％,植株高度增加４．３６％,植株收获期保留青叶数增加１９．４２％,主茎直径增加６．２６％,块根直径增加２．５８％,叶片的叶绿素和蛋白质含量分别提高５．５７％和２５．９６％,叶片光合作用强度提高１５．８６％,而对主茎高度、主茎节数没有明显影响。     

Is gender difference in postnatal thyroid growth associated with specific expression patterns of androgen and estrogen receptors?

Variations in sex steroids bioavailability were linked to the gender difference in the growth of thyroid glands of neonatal rats. In the present study we tested androgen receptor (AR) and estrogen receptor (ER) concentrations by ligand binding assay, and expression of their genes by RT-PCR and Western blot in the thyroid glands of neonatal rats. AR concentration remained elevated from postnatal day (PND) 10 onwards in males, whereas it decreased by PND 20 in females. AR mRNA and protein expressions were higher in males than females, which increased by PND 10, decreased after PND 15 and reached the nadir by PND 20. ER concentration increased by PND 10 and decreased thereafter in both sex. ERα mRNA expression diminished by PND 15 in both sex; while ERβ mRNA decreased by PND 15 to reach the nadir by PND 20 in males, it was augmented by PND 10 in females to reach the peak by PND 15 and diminished by PND 20. ERα protein expression increased by PND 10 and remained elevated till PND 20 in both sex. ERβ protein expression in males increased by PND 10 and decreased by PND 20, while it remained static up to PND 15 and decreased in females. Testosterone stimulated [³H]-thymidine uptake and the expression of IGF-1 and NIS genes in thyrocytes of both sex in vitro, while estradiol stimulated them in females but not in males. We conclude that androgens influence the growth and differentiation of thyrocytes through augmented expression of AR, IGF-1 and NIS in either sex, whereas estrogen imparts the gender difference, which may be at a level beyond the expression of ERs.     

The stimulation by transmitter substances and putative transmitter substances of the net activity of phospholipase A2 of synaptic membranes of cortex of guinea-pig brain.

1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

A Comparison of Preparation Techniques in Taxonomic Studies of Longidorus africanus Merny

A comparison was made of ten different techniques for killing, fixing, and mounting Longidorus africanus Merny for microscopic study. The most satisfactory specimens were those killed by Seinhorst''s method, fixed in FAA and mounted in glycerin by the slow method. Specimens killed by "gentle heat," fixed in FAA and mounted in glycerin were also acceptable as were those killed by hot formalin and mounted in glycerin or processed by Baker''s method. Less satisfactory were: nematodes killed by "gentle heat," fixed in formalin and mounted in glycerin and specimens killed by vapor phase perfusion or by Hopper''s lethal stain, both latter groups were mounted in glycerin after fixation in formalin. Killing with cold formalin, gradual heat (60 C for 15 min), or by storage in distilled water produced poorly defined specimens. Nematodes killed by hot formalin, and processed to glycerin by the slow method, maintained their live dimensions. Reduction in length occurred in specimens killed by cold formalin, by storage, or treated with solutions containing acetic or propionic acids. Nematodes processed by Baker''s method increased in size. Other minor modifications occurred in specimens processed by the different methods. Esophageal definition was best in nematodes killed with formalin, hot or cold. There is no correlation between position of the posterior part of the esophagus and position of the onchiostyle.     

Protective effects of sodium orthovanadate in diabetic reticulocytes and ageing red blood cells of Wistar rats

The reticulocytes and the ageing red blood cells (RBCs) namely young (Y), middle-aged (M) and old RBCs (O) of female Wistar rats from different groups such as control animals (C), controls treated with vanadate (C + V), alloxan-induced diabetic (D), diabetic-treated with insulin (D + I) and vanadate (D + V), were fractionated on a percoll/BSA gradient. The following enzymes were measured-hexokinase (HK), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione-s-transferase (GST), alanine aminotransferase ΜlaAT), aspartate aminotransferase ΜsAT) and arginase in the hemolysates of all the RBCs fractions. Decreases in the activity of HK and AsAT by about 70%, arginase and GSH-Px by 30% in old RBCs were observed in comparison to reticulocytes of control animals. Increases in the activity of GSSG-R by 86%, AlaAT by more than 400% and GST by 70% were observed in old RBCs in comparison to reticulocytes of control animals. Alloxan diabetic animals showed a further decrease in the activities of HK in Y RBCs by 37%, M RBCs by 39% and O RBCs by 32%, GSH-Px activity in Y RBCs by 13%, M RBCs by 20% and O RBCs by 33% and GST activity in Y RBCs by 14%, M RBCs by 42% and O RBCs by 60% in comparison to their corresponding cells of control animals. An increase in the activity of all the enzymes studied was also observed in reticulocytes of diabetic animals in comparison to reticulocytes of controls. The GSSG-R activity was found to be increased in Y RBCs by 49%, M RBCs by 67% and O RBCs by 64% as compared to the corresponding age-matched cells of control animals. The activity of arginase also decreased in Y RBCs by about10%, M RBCs by 20% and O RBCs by 30% in comparison to the age-matched cells of control animals. A decrease in the activity of AsAT in Y and M RBCs by 30%, and O RBCs by 25% was observed in diabetic animals in comparison to the agematched cells of control animals. The activity of AlaAT was found to be decreased by more than 10% in Y and M RBCs and 25% in O RBCs of diabetic animals in comparison to the age-matched cells of control animals. Insulin administration to diabetic animals reversed the altered enzyme activity to control values. Vanadate treatment also reversed the enzyme levels except for that of GST in old cells     

A new way to monitor by-pass restorations of electron transport in inhibited chloroplasts by cyclic electron flow cofactors--a study by modulated fluorimetry.

Inhibition of electron transport in broken chloroplasts by DBMIB, under light-limiting conditions, is shown to be bypassed by PMS in a manner similar to the known effects of the phenylenediamine derivatives TMPD and DAD. These bypasses were demonstrated and further studied by modulated fluorimetry, monitoring DBMIB inhibition by the shift of the steady-state fluorescence towards the Fm level and the release of inhibition by a reverse shift together with establishment of a quenching effect by background far-red light. Comparative studies were also made with electron transport blocked by DCMU or BNT. A weak bypass by TMPD and a weaker one by PMS of the block created by DCMU was observed by modulated fluorimetry. The block created by BNT is similarly shown to be bypassed by TMPD but hardly or not at all by PMS. Bypass effects persisted even in the presence of ascorbate. It appears that, following reduction of the different cofactors by ascorbate in the stroma side, illumination caused the accumulation of a pool of oxidized cofactor molecules in the lumen, which is able to mediate electron transport between reduced plastoquinone and plastocyanin or P-700. The existence and the size of this pool were found to depend largely on the internal pH at the lumen, presenting an artificial system in which electron flow is controlled by the lumenal pH. The bypassing electron transport in the presence of DBMIB presumably avoids the participation of the cytochrome b6f complex. During its occurrence, there is also a strong imbalance in the activities of the two photosystems for linear electron flow, in favor of PS II. These experiments may thus serve to establish an in vitro model system for a future investigation of effects related to changes in the imbalance between the two photosystems and its regulation. Furthermore, this experimental system may also be utilized to study the role of the internal lumenal pH in control of photosynthesis.     

Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN

We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITC-albumin by the cells was time and temperature dependent and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-beta-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed.     

O2 transport in cerebral microregions (mathematical simulation)

The effects of the circulation rate in capillaries, the intensity of O2 consumption by nerve cells and the capillary network density on the O2 tension distribution in the cerebral cortex have been studied, utilizing a mathematical model simulating actual neuron-capillary relationships. The model has been written as a system of equations in partial derivatives, its solution obtained by the net-point method. Regulatory variations of the capillary circulation rate in certain cerebral microregions have been shown to ensure similar changes in oxygen supply throughout the region. A drop of the pO2 level in a cerebral microregion with a rising O2 consumption by nerve cells is shown to be due, by 75 percent, to the increase of O2 consumption and by 25 percent, to the lower pO2 in the capillaries. Conversely, an increase in pO2 in microregions resulting from a lower O2 consumption by neurons is due by 75 percent, to a pO2 rise in capillaries and by 25 percent, at the expense of an O2 consumption decrease. In cerebral regions differing in capillary network density by 20 percent, changes in the conditions for oxygen supply to tissue are due by 1/3 to pO2 variations in the capillaries and by 2/3 to alterations in the diffusion distances.     

Effect of thiamine, riboflavin and ascorbic acid on tetracycline, erythromycin and levomycetin activity]

The effect of antibiotics was estimated by inhibition of the protein increase in the broth culture of Staph, aureus during incubation at a temperature of 37 degrees for 18 hours. In some experiments preincubation of the antibiotic solutions with the vitamins for 2 hours at light and in dark was used. The antibiotic concentrations in gamma per 1 ml were equal to those of the vitamins. In the experiments with tetracycline and 2-hour preincubation at light the antibiotic in a concentration of 0.1gamma ml inhibited for certain the protein increase by 58.9%, in combination with thiamin it inhibited the protein by 60 per cent and in combination with ascorbic acid by 59%. Riboflavin lowered the activity of tetracycline to a value not differing for certain from the control one. In the experiments with preincubation in dark tetracycline inhibited the protein increase by 55.2%, in combination with thiamin it inhibited the protein increase by 50.5%, in combination with riboflavin by 53% and in combination with ascorbic acid by 57.2%. Erythromycin in a concentration of 0.03gamma/ml when preincubated at light inhibited the protein increase by 48.8% and in combinations with thiamin, riboflavin or ascorbic acid by 23, 27, 47.2% respectively. When preincubated in the darkness erythromycin alone inhibited the protein increase by 47.8% and in combinations with thiamin, riboflavin or ascorbic acid by 32.5, 51.1 or 49.8% respectively. The above vitamins has no effect on levomycetin activity.     

研究荧光标记BCAA及AAA在大鼠体内代谢

使用DNS-Cl标记BCAA及AAA等六种氨基酸,观察这些氨基酸在大鼠小肠的吸收,血液的清除以及肝脏、肌肉、肾脏、脑等器官的摄取及排空情况。结果:小肠在30分钟开始吸收,4小时部分排空;血液在4小时开始清除,16小时清除完毕;肝脏、肌肉、肾脏、脑组织等,都在30分钟全部或部分摄取,但各器官对不同氨基酸,排空的时间不尽相同。     

A prothrombinase complex of mouse peritoneal macrophages

Addition of prothrombin to mouse peritoneal macrophages in vitro resulted in the formation of a thrombin-like enzyme, as demonstrated by use of the luminogenic peptide substrate S-2621. The prothrombinase activity was sedimented by high-speed centrifugation following homogenization of the cells and was abolished by treatment of the cells with the nonionic detergent Triton X-100 at 0.02% concentration. Moreover, the activity was drastically reduced by maintaining cultures in the presence of warfarin and, presumably due to competitive substrate inhibition, by adding S-2222, a chromogenic peptide substrate for Factor Xa. These findings suggest that prothrombin cleavage is catalyzed by Factor Xa at the macrophage surface. The generated thrombin was inhibited by antithrombin, and this reaction was accelerated by heparin with high affinity for antithrombin but not by the corresponding oligosaccharides composed of 8-14 monosaccharide units. Such oligosaccharides which are capable of accelerating the inactivation of Factor Xa by antithrombin, inhibited thrombin formation from prothrombin in the macrophage cultures, presumably by promoting inactivation by antithrombin of Factor Xa in a prothrombinase complex. Activation of the macrophage coagulation system, as proposed to occur in certain inflammatory conditions, thus may be modulated at various levels by heparin, or heparin oligosaccharides, released from mast cells.     

Homocarnosine content and homocarnosine-carnosine synthetase activity in brain areas of hyperoxic rats

The homocarnosine content and homocarnosine synthetase activity were studied in the brain of rats in normal state and under hyperoxia. The homocarnosine content is higher in phylogenetically old brain areas as compared with that in the cerebral hemispheres. Its nonuniform distribution in the brain is associated with different activity of homocarnosine-carnosine synthetase in the corresponding brain areas. At the preconvulsive stage of oxygen poisoning the homocarnosine content in all the brain areas does not change, the homocarnosine-carnosine synthetase activity is 32% lower. At the convulsive stage of hyperoxia the homocarnosine amount in the cerebral hemisphere decreases by 33%, in the midbrain and diencephalon -- by 70, in the medulla oblongata -- by 60, in the cerebellum -- by 58%. The decrease in the homocarnosine content correlates with that in the activity of homocarnosine-carnosine synthetase in the corresponding brain areas; in the cerebral hemispheres -- by 33%, in the midbrain and diencephalon -- by 50, in the medulla oblongata -- by 49, in the cerebellum -- by 40%.     

A novel potent inhibitor of inducible nitric oxide inhibitor, ONO-1714, reduces intestinal ischemia-reperfusion injury in rats.

The overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of intestinal injury induced by ischemia-reperfusion. The aim of the present study was to examine the effect of selective iNOS inhibition by a cyclic amidine analogue, ONO-1714, on reperfusion-induced small intestinal injury and inflammation in rats. Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. The luminal nitrite concentration in the small intestine was measured by Griess reaction and the iNOS mRNA expression by RT-PCR. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers and by the histological findings. The rats which were killed after ischemia-reperfusion had increased luminal concentrations of nitrite and iNOS mRNA expression, in addition to severe intestinal inflammation characterized by significant increases in myeloperoxidase activity, a marker of neutrophil infiltration, and by the mucosal content of CINC-1 cytokine, a neutrophil chemotactic cytokine. Administration with ONO-1714 significantly inhibited the luminal NO production. Reperfusion after 30-min ischemia resulted in an increase in luminal protein and hemoglobin concentrations, with levels reaching a maximum after 60 min of reperfusion. In contrast, pre-treatment with ONO-1714 2h before the ischemia inhibited the increases in luminal protein and hemoglobin concentration in a dose-dependent manner (0.001-0.1mg/kg). The contents of the thiobarbituric acid-reactive substances (a marker of oxidative lipid peroxidation) were significantly increased by ischemia-reperfusion, and this increase was reduced by ONO-1714. After reperfusion, the increase in tissue-associated myeloperoxidase activity, an index of neutrophil infiltration, was significantly inhibited by pre-treatment with ONO-1714. ONO-1714 also inhibited increases in intestinal CINC-1 protein and mRNA expression, as determined by ELISA and RT-PCR, respectively. In conclusion, the improvement of reperfusion-induced intestinal injury by ONO-1714 suggested that an excess of NO, produced by iNOS, may have contributed to the initiation/amplification of intestinal inflammatory injury by various mechanisms, including nitrosative and oxidative damage as well as the enhancement of inflammatory cytokine release.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

Induction of the non-selective mitochondrial pore in lymphoid cells. 1. Permeabilized rat thymocytes.

The opening of the cyclosporin-sensitive pore in the inner membrane of mitochondria in rat thymocytes was studied. In thymocytes with digitonin-permeabilized plasma membrane, the mitochondrial pore was induced by Ca2+ overload, by uncoupling, by oxidation or cross-linking of membrane dithiols, and by atractyloside, a specific inhibitor of the adenine nucleotide transporter. Pore opening was prevented by cyclosporin A (CsA) and by its non-immunosuppressive analog MeVal-CsA. The sensitivity of the pore to CsA was decreased by atractyloside and practically disappeared when it was added in combination with uncoupler. The main properties of the pore in mitochondria from thymocytes and from hepatocytes are the same. Release of Ca2+ from thymocyte mitochondria induced by uncoupling is mediated by a specific uniporter and by the pore with similar rates.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.