Alphaxalone, a neurosteroid anaesthetic, increases the activity of the glutamate transporter type 3 expressed in Xenopus oocytes

Glutamate transporters may be important targets for anaesthetic action in the central nervous system. The authors investigated the effects of alphaxalone, an intravenous neurosteroid anaesthetic, on the activity of glutamate transporter type 3 (EAAT3). EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after applying l-glutamate (30 μM) in the presence or absence of alphaxalone. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Results are presented as means ± S.E.M. l-Glutamate induced inward currents in EAAT3 expressing oocytes, and these currents were dose-dependently increased by alphaxalone. Alphaxalone at 0.01 to 3 μM significantly increased the inward currents. In addition, the treatment of oocytes with phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the transporter currents (1.0 ± 0.2 to 1.4 ± 0.2 μC; P < 0.05). However, treatment with PMA plus alphaxalone did not increase responses further as compared with PMA or alphaxalone alone. Furthermore, pretreatment of oocytes with chelerythrine or staurosporine, two PKC inhibitors, did not affect basal transporter currents, but did significantly reduce alphaxalone-enhanced EAAT3 activity; whereas oocytes pretreated with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, showed significant reductions in basal and alphaxalone-enhanced EAAT3 activities. The above results suggest that alphaxalone enhances EAAT3 activity and that PKC and PI3K are involved in this effect.     

Increased levels of phorbin, c-myc, and ornithine decarboxylase RNAs in human colon cancer.

Our previous work on protein kinase C (PKC) and colon cancer has shown altered levels of PKC activity in human colon tumors, as well as activation of PKC by colon tumor promoters such as bile acids. To understand further the role of PKC in colon carcinogenesis, we analyzed the expression of phorbin, a gene induced by PKC activation, in a series of different stages of human colon tumors. As shown by northern blot analyses of poly (A)+ RNA, higher levels of phorbin RNA were seen in 26 colon tumor samples than in their adjacent normal colonic mucosa. There also appeared to be a correlation between the abundance of phorbin RNA in the tumors and the extent of invasion (tumor-to-normal tissue phorbin RNA ratio = 4.2, 8.0, and 11.9 for Dukes' A, B, and C, respectively). Phorbin RNA was also abundant in a human colon cancer line (HT29). We also examined the expression of other mitogen-responsive genes (c-myc, ODC, and beta-actin) in a set of 19 colon tumor samples. All tumors displayed significant (mean 3.8-fold) increases in the level of c-myc RNA compared with their adjacent normal colonic mucosa. About 47% and 16% of these tumor samples also showed increased levels of ODC (mean 3.1-fold) and beta-actin (mean 1.6-fold) RNA, respectively. The increased levels of c-myc, ODC, and beta-actin RNA did not correlate with the extent of tumor invasion. Taken together, these results demonstrate that human colon tumors usually display increased levels of both phorbin and c-myc RNAs. The marked increases in phorbin RNA suggest that this could serve as a useful biomarker in studies on human colon cancer.     

Calcium-activated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes

Phosphoinositide-linked transmembrane signaling in the brain involves calcium-activated, phospholipid-dependent protein kinase (protein kinase C), but little is known about the glial contribution to this system. We observed that phosphorylation of several proteins in a cytosal fraction of rat astrocytes in primary culture was increased by the addition of calcium and phosphatidylserine. These agents also stimulated phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. Addition of diacylglycerol, an activator of protein kinase C, further increased histone phosphorylation, whereas polymyxin B, an inhibitor of protein kinase C, blocked the stimulatory effect of calcium and phosphatidylserine. Based on enzyme units per mg protein, the activity of protein kinase C in astrocytes appears similar to that in whole brain cytosol. These results indicate that astrocytes display protein kinase C activity and suggest that the glial enzyme may be an important component of the receptor-linked phosphoinositide response system in the brain.     

Interacting with HBsAg compromises resistance of jumping translocation breakpoint protein to ultraviolet radiation-induced apoptosis in 293FT cells

Jumping translocation breakpoint protein (JTB) is suppressed in many cancers, implying it plays a role in the neoplastic transformation of cells. In order to explore the role of JTB in the carcinogenesis of liver, we used mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal to verify the interaction between HBs and JTB. According to the results, HBs interacts with JTB. In addition, we further determined that S region within HBs is sufficient for binding JTB. Overexpression of JTB conferred resistance to apoptosis induced by ultraviolet radiation, whereas this effect was compromised by the co-overexpression of HBs.     

Protein kinase C and Gi-protein mediated modulation of cAMP production in different stages of the rat seminiferous epithelium

The modulatory role of protein kinase C (PK-C)- and G_i-protein-mediated signal transduction systems was studied in the cyclic variation of follicle-stimulating hormone (FSH)-stimulated cAMP production of rat seminiferous tubules. FSH (Metrodin, Serono, 30 mg/1) stimulated cAMP production 10-fold (p < 0.01) in a 3 h incubation of 5 mm segments of seminiferous tubules of stages II–VI of the epithelial cycle, but only 2-fold (p < 0.01) in stages VII–VIII. The PK-C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nmol/1) suppressed the FSH effect on cAMP output by 50–70% (p < 0.01) in stages II–VI, but had no effect in stages VII–VIII. If the tubular segments were preincubated for 3 h in the presence of pertussis toxin (PT, 100 μg/1), the FSH-stimulated cAMP production of stages VII-VIII increased by 100–200% (p < 0.01), and now they also became responsive to the TPA suppression. Conversely, no effect of PT was observed in stages II–VI. Cholera toxin (CT, 100 μg/1) and forskolin (Fk, 100 μmol/1) nearly similarly stimulated the cAMP production in both stages studied (about 10-fold, p < 0.01), and TPA and PT potentiated the effects in a non-additive fashion. In conclusion, both G_i-protein and PK-C-mediated mechanisms modulate cAMP production of rat seminiferous tubules. A clear cyclic variation can only be demonstrated in FSH-stimulated cAMP production, but not if the G_s-protein or adenylate cyclase are directly stimulated. Upon FSH stimulation, the low cAMP production in stages VII–VIII is mainly due to the G_i-protein-mediated inhibition. In contrast, the absence (or non-function) of this inhibition mechanism explains the brisk cAMP response to FSH in stages II–VI. PK-C activation suppresses FSH-stimulated cAMP production only if it is not inhibited by the G_i-protein-mediated mechanism (stages II–VI), probably by inhibiting the FSH-receptor-G_s-protein association. It also increases CT and Fk-stimulated cAMP production, in this case inactivating the G_i-protein.     

Characterization of a glucocorticoid-sensitive hippocampal protein

Increased synthesis of a rat hippocampal protein with an apparent molecular weight (Mr) of 35,000 Da occurs in response to elevation of serum corticosterone levels. Subcellular fractionation has localized this protein in the cytosol. Two-dimensional gel electrophoresis indicated that this protein has an isoelectric point (IEP) of 6.6. A similar protein in liver has a slightly higher Mr and an IEP of 6.8. Increased synthesis of one additional hippocampal protein with an Mr of 46,000 Da and an IEP of 6.2 and of two other liver proteins, one with an Mr of 53,000 Da and an IEP of 6.2 and the other with an Mr of 45,000 Da and a range of IEPs from 8.7 to 7.8, was also seen after injection of corticosterone into rats. One possible identity of the 35,000 Da protein is glycerol 3-phosphate dehydrogenase (GPDH), based upon the reported Mr and IEP of this enzyme. The 35,000 Da hippocampal protein co-eluted from a gel filtration column with GPDH activity. No alteration of hippocampal GPDH activity was seen in intact rats 4 or 24 h after injection of either corticosterone or the type II receptor-specific agonist RU 28362. However, daily administration of corticosterone to rats beginning 10 days after adrenalectomy returned hippocampal GPDH activity to normal values after 2-3 days. In contrast, synthesis of the 35,000 Da protein was maximally increased 4 h after a single injection of steroid and not elevated at later times.     

细胞角蛋白8与肿瘤

细胞角蛋白8(CK8)是一种中间丝蛋白,属于细胞骨架蛋白家族.研究表明,CK8表达或翻译后修饰异常将影响其活性,是相关肿瘤发生发展的重要原因之一.对CK8在肿瘤发生发展中作用的深入研究,可以为肿瘤的预防及治疗提供帮助.     

Effects of cadmium on human platelet reactions

Human platelets incubated with Cd²⁺ took up the cation slowly, and the uptake was speeded up by ionophore A23187. The capacity of human platelets to accumulate Cd²⁺ was large, equivalent to 10 nmol Cd²⁺ per mg protein. The effects of Cd²⁺ on protein phosphorylation and serotonin release of human platelets were studied. Washed platelets incubated with Cd²⁺ showed a general increase in protein phosphorylation concurrent with a slow release of serotonin. In the presence of ionophore A23187, however, Cd²⁺ had a biphasic effect on protein phosphorylation: stimulatory at low and inhibitory at high Cd²⁺ concentrations. The phosphorylation of two proteins with molecular masses close to 43 and 20 kDa was more sensitive to the inhibitory effect of Cd^2&#x002B;, and under similar conditions, the primary effect of Cd²⁺ on serotonin release was inhibitory, although at lower Cd²⁺ concentrations a slight stimulation was noted. Thrombin increased the phosphorylation of several proteins, and a prior incubation with Cd²⁺ further augmented that of a 20 kDa protein, but this treatment did not affect thrombin-induced serotonin release.     

Antiinflammatorische Therapie in der Sepsis

A systemic inflammation with the release of multiple cytokines plays an important role in the pathophysiology of sepsis. During the last years, several anti-inflammatory substances have been investigated with respect to their effects on mortality in patients with sepsis. However, only the antibody fragment of the TNFα binding antibody afelimomab and the recombinant human activated protein C (drotrecogin α [acivated]) were capable of improving the outcome in controlled studies with large sample sizes. The possible administration of these substances should be restricted to patients who meet the inclusion criteria of these studies. In particular, the tight time window, which usually ends 24 h after the onset of sepsis, should be taken into consideration before starting an anti-inflammatory medication. In addition to the anti-inflammatory treatment, the control of the infectious focus and an aggressive hemodynamic stabilization must not be neglected. Ibuprofen, interleukin-1 receptor antagonists and soluble TNFα-receptors as well as high dosages of corticosteroids and antithrombin III do not have a place in the anti-inflammatory treatment of sepsis.     

中国仓鼠V79细胞系的oua~R和oua~S细胞蛋白质组分的双向电泳比较

作者用聚丙烯酰胺凝胶双向电泳,对中国仓鼠V79细胞系的乌本苷抗性突变型(oua~R)和野生型(oua~S)细胞的蛋白质组分进行比较,选择20个高度重复、分布均匀的肽斑作为参考标记,发现一些多肽在其位置、大小及密度上表现出差异,值得注意的是4小区(pI/MW为5.0/28～30kd)和6小区(pI/MW为6.3/39kd)的变化明显,提示这些多肽可能对V79细胞的oua~R是特异的,也许可作为oua~R细胞的特征性遗传标记。