Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

DTI定量参数对脑胶质瘤的诊断价值及其与血管内皮生长因子、细胞核增殖相关抗原的关系

目的:探讨扩散张量成像(DTI)定量参数对脑胶质瘤的诊断价值及其与血管内皮生长因子(VEGF)、细胞核增殖相关抗原(Ki-67)的关系。方法:选取2014年6月到2017年6月期间在我院接受治疗的90例脑胶质瘤患者,根据病理分级的不同分为中低级别组(n=46)和高级别组(n=44),比较两组患者表观扩散系数(ADC)值、各向异性分数(FA)值、相对表观扩散系数(rADC)值、相对各向异性分数(rFA)值、VEGF和Ki-67的阳性率,分析ADC值、FA值、rADC值、rFA值与VEGF、Ki-67表达的相关性。结果:高级别组的ADC值、FA值、rADC值和rFA值低于中低级别组(P0.05)。高级别组病理组织中VEGF、Ki-67的阳性表达率高于中低级别组(P0.05)。经Spearman相关分析显示,ADC值、FA值、rADC值和rFA值与VEGF、Ki-67的表达水平均呈负相关(P0.05)。结论:DTI定量参数与脑胶质瘤病理分级和VEGF、Ki-67的表达水平密切相关。     

Expression of the small heat shock protein (hsp) 27 in human astrocytomas correlates with histologic grades and tumor growth fractions

Summary 1. Cellular expression and distribution of the stress response small heat shock protein 27 (hsp27) in 39 high-grade astrocytomas (27 glioblastoma multiformes, 12 anaplastic astrocytomas) and in 27 low-grade astrocytomas (grade I–II) were analyzed immunohistochemically.2. The correlation between hsp27 expression and tumor growth fractions of the astrocytomas was examined following Ki-67 immunostaining.3. The hsp27 staining was cell cytoplasmic. The hsp27 immunopositive rate was significantly higher in high-grade astrocytomas; the rates were 74% for glioblastomas, 58% for anaplastic astrocytomas, and 37% for low-grade astrocytomas. The small and large tumor cells, especially in glioblastomas, multinucleated tumor giant cells, tumor cells in the pseudopalisading and necrotic areas, cells of the microvascular endothelial proliferations, and tumor vascular smooth muscles were usually hsp27 positive. The mean percentage of hsp27-positive cells was significantly higher in the glioblastomas alone and in the combined high-grade astrocytomas, compared to the low-grade, and in recurrent rather than in primary high-grade astrocytomas.4. The high-grade astrocytomas had a highly statistical significant Ki-67 labeling index. The Ki-67 labeling indices were significantly higher in the hsp27-positive than the hsp27-negative astrocytomas, irrespective of the histological grade. In the high-grade astrocytomas with a Ki-67 labeling index of five and above, 81% of those tumors were hsp27 positive.5. Thus, a large number of human astrocytomas express hsp27, and hsp27 expression correlates with histological grades of astrocytoma and with tumor growth fractions. This being the case, hsp27 is likely to have a role in the growth of human astrocytomas.     

Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by munc-18

Neuronal cdk5 can phosphorylate certain lys-ser-pro (KSP) motifs of neurofilaments and tau protein in the nervous system. We have immunoprecipitated the cdk5 from rat brain using a polyclonal antibody raised against the C-terminus of cdk5. The immunoprecipitate has phosphorylated a KSPXK peptide analog of NF-H, as well as histone H1 and a bacterially expressed rat NF-H protein. The kinase activity was inhibited by staurosporine, isopentanyladenine and olomoucine in a dose dependent manner. Kinetic studies indicated Ki values of 39 nM, 38 μM and 8 μM, respectively for staurosporine, isopentanyladenine and olomoucine. The inhibition by staurosporine was non-competitive with respect to phosphoryl acceptor substrates. Western blot analysis of the immunoprecipitate showed both cdk5 and p67 (munc-18), a putative regulator molecule of the kinase. Addition of p67 fusion protein enhanced the kinase activity of the immunoprecipitate by 60% above the basal activity. P67 elevated Ki values for both staurosporine and olomoucine. The degree of inhibition at high concentrations of these inhibitors was unaltered by exogenous p67 indicating a lack of competitive interactions with p67. The high affinity of staurosporine for cdk5 suggests that cdk5 may be one of the targets for the neurotropic effect of staurosporine.     

Postresuscitation Changes in Brain Free Radical-Mediated Processes and Nitric Oxide Synthase Activity in Rats: Effects of Individual Behavior in "Emotional Resonance” Test

Effects of 7-min cardiac arrest and individual behavior on free radical-mediated processes and nitric oxide synthase (NOS) activity was evaluated in brains of male Wistar rats one hour and one week after resuscitation. "Emotional resonance test was used for the behavioral selection of rats. The test includes factors of significance for rats: the choice between large and lighted or small and dark space as well as signals of pain of another rat. Free radical generation (using chemiluminescence method), superoxide scavenging/generating activity, substances reacting with 2-thiobarbituric acid and NOS activity (by measuring mononitrosyl iron complex of NO with diethyl dithiocarbamate and endogenous brain Fe²⁺ by electron spin resonance spectroscopy) were determined in cerebral cortex, cerebellum and hippocampus. Cardiac arrest induced oxidative stress accompanied by the loss of NOS activity, as well as compensatory changes of free radical-mediated processes in cerebral cortex. Oxidative stress was also evident in cerebellum and, to a lesser extent, in hippocampus. Most of neurochemical differences between behavioral groups were induced by cardiac arrest. These differences were global, related to a specific brain region or became apparent in cerebral lateralization of biochemical indices.     

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.     

Uptake of radiolabeled copper from portal blood containing fructose or glucose

The present investigation was designed to study the uptake of⁶⁷Cu when administered directly, into the portal vein, along with either functose or glucose, by the liver and extrahepatic tissues. Following weaning, male Sprague-Dawley rats were fed for 3 wk either commercial laboratory ration (chow) or semipurified diets deficient in Cu (0.6 ppm) or supplemented with Cu (6.0 ppm) and containing 62% carbohydrate as either fructuse or cornstarch. After an overnight fast, a single dose of rat plasma (0.1 mL) containing fructose or glucose extrinsically labeled with⁶⁷Cu was injected directly into their portal vein. Although not always statistically significant, rats fed chow retained more radioactivity in the liver and several extrahepatic tissues when⁶⁷Cu was administered with fructose than with glucose. Regardless of Cu status, rats fed diets containing fructose retained more radioactivity in extrahepatic tissues than rats fed starch. There was an increased uptake of⁶⁷Cu by the liver, blood, muscle, and fat pad when fructose as compared to glucose was injected in combination with the isotope. These data strongly suggest that Cu requirements or utilization are greater when fructose is the main dietary carbohydrate. The results may also in part explain the reason for the increased severity of Cu deficiency in rats fed fructose.     

Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid