Early induction of neuronal lipocalin-type prostaglandin D synthase after hypoxic-ischemic injury in developing brains

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is up-regulated in oligodendrocytes (OLs) in mouse models for genetic neurological disorders including globoid cell leukodystrophy (twitcher) and GM₁ and GM₂ gangliosidoses and in the brain of patients with multiple sclerosis. Since L-PGDS-deficient twitcher mice undergo extensive neuronal death, we concluded that L-PGDS functions protectively against neuronal degeneration. In this study, we investigated whether L-PGDS is also up-regulated in acute and massive brain injury resulting from neonatal hypoxic-ischemic encephalopathy (HIE). Analysis of brains from human neonates who had died from HIE disclosed that the surviving neurons in the infarcted lesions expressed L-PGDS. Mouse models for neonatal HIE were made on postnatal day (PND) 7. Global infarction in the ipsilateral hemisphere was evident at 24 h after reoxygenation in this model. Intense L-PGDS immunoreactivity was already observed at 10 min after reoxygenation in apparently normal neurons in the cortex, and thereafter, in neurons adjacent to the infarcted area. Quantitative RT-PCR revealed that the L-PGDS mRNA level of the infarcted hemisphere was 33-fold higher than that of the sham-operated mouse brains at 1 h after reoxygenation and that it decreased to the normal level by 24 h thereafter. Furthermore, in both human and mouse brains, many of L-PGDS-positive cells were also immunoreactive for p53; and some of these expressed cleaved caspase-3. The expression of L-PGDS in degenerating neurons implies that L-PGDS functions as an early stress protein to protect against neuronal death in the HIE brain.     

阿霉素对兔心肾损伤与一氧化氮合酶表达

目的:探讨阿霉素(Adr)对心和肾损伤与一氧化氮合酶(NOS)表达。方法:用治疗剂量阿霉素静脉注射法建立兔心、肾损伤模型组,测定兔心输出量(CO)、左心室收缩压(LVSP)、左心室舒张末压(LVEDP):取心和肾组织进行HE染色及超薄切片染色;NADPH组化染色并进行图像分析,观察动物心和肾组织中ALP、Ca~(2+)-ATP酶和 NOS的变化。结果:实验组CO、LVSP均明显低于对照组,LVEDP明显高于对照组;实验组心和肾组织的超微结构受损伤,Ca~(2+)-ATP酶活性明显下降,而NOS表达明显上调。结论:治疗剂量阿霉素能够降低心和肾组织中Ca~(2+)-ATP酶的活性和上调NOS的表达,是导致心和肾组织损伤的重要原因。     

Chromogranin A activates diverse pathways mediating inducible nitric oxide expression and apoptosis in primary microglia

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimer's, Pick's and Parkinson's diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimer's disease.     

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18

Rat Kupffer cell (KC)-mediated cytotoxicity against both thesyngeneic hepatoma cell line AH70 and hepatocytes was evaluatedby changes in mitochondrial function, and the possible roleof ICAM-1/CD18 in the interaction between the cells was studied.Rhodamine 123 fluorescence, a marker of the mitochondrial membranepotential, decreased in AH70 cells after co-culture with KC,while that in hepatocytes was unchanged by co-culture. Thisdecrease was blocked by anti-ICAM-1, anti-CD18 and the Inhibitionof nitric oxide synthesis. Cytometric studies demonstrated thatICAM-1 expression on AH70 cells increased after addition ofIFN-, IL-1ß, tumor necrosis factor (TNF)- or KC, whilein hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatmentinhibited the increase in ICAM-1 expression and the decreasein rhodamine 123 fluorescence on AH70 cells after co-culturewith KC. CD18 on KC was increased only after co-culture withAH70. TNF- but not IFN- was detected in the supernatant of co-culturebetween KC and AH70 cells, and this production was partiallyinhibited by anti-ICAM-1 and anti-CD18. The activity of Induciblenitric oxide synthase in Kupffer cells and the levels of nitritesand nitrates in the co-culture supernatant increased over time,and this increase was attenuated either by addition of NO synthesisinhibitors, anti-ICAM-1 or anti-CD18. These results indicatethat the rat KC causes mitochondrial dysfunction in cancer cellsvia the production of NO and cell-to-cell adhesion via ICAM-1/CD18has an Important role in this cytotoxic process.     

Sites of arginine synthesis and urea production along the nephron of a desert rodent species,Meriones shawi

The distribution of arginine synthase and arginase activities along the successive nephron segments ofMeriones kidney was measured in vitro with single tubule enzymatic microtechniques making use of eitherl-[ureido-¹⁴C] Citrulline (0.108 mM) orl-[guanidion-¹⁴C]arginine (0.2 mM) as the respective substrates. Arginase activity (fmol urea formed per min per mm of tubule) was very low (5–25 fmol.min^–1.mm^–1) in most nephron segments including the early portions of proximal convoluted tubules (early PCT). It increased progressively after 3 mm of the PCT to reach a value of 200 fmol.min^–1.mm^–1 in the cortical portion of the straight proximal tubule (CPST), with a further increase, along the pars recta, of up to 250 fmol.min^–1.mm^–1 in the outer medullary portion (OSPST). In addition, arginase activity in OSPST and the adjacent descending thin limb (DTL) was higher in juxtamedullary nephrons (JN) than in the corresponding portions of superficial nephrons (SN). Arginine synthase activity (fmol arginine formed per mm of tubule per min) was present in proximal tubules exclusively, with a gradient decreasing along the PCT (about 600 fmol.min^–1.mm^–1 in the 1st mm, 65 fmol.min^–1.mm^–1 in CPST and 30 fmol. min^–1. mm^–1 in OSPST). It has been checked that CPST and OSPST (where the two enzyme systems are present) are able to convert citrulline directly into urea with a yield of 65%. It is suggested that: (1) in early PCT cells, arginine synthase activity permits the conversion of the reabsorbed citrulline into arginine (which then diffuses towards blood vessels); and (2) in pars recta cells, arginase activity results in a net entry of arginine across the basolateral membranes and in a net exit of the formed urea into the tubular fluid, if the permeability to urea of luminal membranes is greater than that of basolateral membranes. Such a mechanism of urea secretion might contribute to the maintenance of urea recycling in the medulla and, thereby, participate in the process of concentrating the urine.     

施万细胞和一氧化氮合酶抑制剂对大鼠脊髓损伤后背核神经元存活及其轴突再生的影响

目的探讨施万细胞(SCs)和一氧化氮合酶(NOS)抑制剂L-NNA联合应用能否促进脊髓损伤后背核神经元存活及其轴突再生。方法20只成年大鼠分为对照组、SCs组、L-NNA组和SCs L-NNA组。在SCs L-NNA组动物T11脊髓段半横断后在损伤处植入施万细胞，术后腹腔内注射L-NNA。结果脊髓半横断后30d，对照组L1脊髓段损伤侧背核神经元数量减少，其胞体皱缩，NOS表达阳性。SCs组存活的神经元增加的同时其NOS表达增强，胞体也发生皱缩。L-NNA组和SCs L-NNA组存活的神经元也增加，但NOS表达降低，其胞体皱缩得到改善。各组中仅在SCs L-NNA组L1脊髓损伤侧背核观察到有被FG标记的神经元胞体，提示其再生轴突穿越损伤区到达头端脊髓组织。结论SCs和L-NNA都可促进脊髓半横断背核受损伤神经元的存活；两者联合应用能更好地促进受损伤背核神经元存活及其轴突再生。     

Expression of a NOS transgene in dystrophin-deficient muscle reduces muscle membrane damage without increasing the expression of membrane-associated cytoskeletal proteins

Muscular dystrophy that is caused by mutation of the membrane-associated, cytoskeletal protein called dystrophin, is accompanied by loss of a dystrophin-associated protein complex (DPC) that includes neuronal nitric oxide synthase (nNOS). Previous work showed that expression of a nNOS transgene in the dystrophin-deficient, mdx mouse greatly reduces muscle membrane damage. In this investigation, we test whether expression of a nNOS transgene in wild-type or mdx muscle increases expression of DPC proteins, or functionally related proteins in the integrin complex that are upregulated in dystrophin-deficiency, or affects expression of the dystrophin homolog, utrophin. Many members of the DPC are enriched in Western blots of cell membranes isolated from NOS transgenic muscle, compared to wild-type. Similarly, alpha7-integrin and the associated cytoskeletal proteins talin and vinculin are increased in NOS transgenic, non-dystrophic muscle. However, utrophin expression is unaffected by elevated NOS expression in healthy muscle. A similar trend in mRNA levels for these proteins was observed by expression profiling. Analysis of membrane preparations from mdx mice and NOS transgenic mdx mice shows that expression of the NOS transgene causes significant reductions in utrophin, talin, and vinculin. Expression profiling of mRNA from mdx and NOS transgenic mdx muscles also shows reduced expression of talin. Immunohistochemistry of mdx and NOS transgenic mdx muscle indicates that reduction in utrophin in NOS transgenic mdx muscle results from a decrease in regenerative fibers that express high levels of utrophin. Together, these findings indicate that the NOS transgene does not reduce dystrophinopathy by increasing the expression of compensatory, structural proteins.     

Protective effect of retinoic acid on interleukin-1 beta-induced cytotoxicity of pancreatic beta-cells

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.     

施万细胞对大鼠脊髓半横切后背核神经元存活及其表达NOS的影响

目的：探讨移植的施万细胞对大鼠脊髓半横切后背核神经元存活及其表达NOS的影响。方法：50只成年SD大鼠被分为实验组和对照组。在胸11脊髓段半横切后立即在损伤处移植入施万细胞。结果：脊髓半横切后，15d和25d对照组L1脊髓段损伤侧背核神经元的存活数均比未损伤侧的明显减少。存活的神经元胞体出现明显皱缩，有些神经元呈现NADPH－d阳性。15d和25d施万细胞组L1脊髓段损伤侧背核神经元的存活数则比同期对照组的明显增加，表达NOS的存活神经元数也随之增多。但存活的神经元胞体仍然是皱缩的。结论：移植的SCs可促进受损伤的背核神经元存活及其表达NOS，但不能阻止其胞体出现皱缩。     

金属硫蛋白对培养神经细胞迟发性损伤的作用和一氧化氮的表达

本文以新生大鼠原代培养皮质神经元为实验材料，造成迟发性神经元损伤模型．在不同时程内，测试培养液中的细胞乳酸脱氢酶漏出量和用还原型尼克酰胺腺嘌呤二核着苷酸脱氢酶染色反应，观察培养细胞中一氧化氮合酶的表达水平．结果表明，缺血组在缺血与再灌流中，细胞乳酸脱氢酶漏出量明显高于对照组，差异显著（Ｐ＜０．００１）．一氧化氮合酶阳性神经元数量在缺血组的缺血时即明显高于对照组（Ｐ＜０．０１），再灌流后一氧化氮合酶表达仍然强烈，与对照组相比有显著差异（Ｐ＜０．０１）．当损伤细胞再灌流时，同时加入金属硫蛋白后，显示一氧化氮合酶表达减弱，和缺血组相比有显著差异（Ｐ＜０．０５～０．００１），细胞乳酸脱氢酶漏出量在再灌流后的早期近似于对照组．结合文献和本实验结果提示：在迟发性神经元损伤形成过程中一氧化氮起着重要作用；金属硫蛋白对脑缺血后迟发性神经元损伤有一定保护作用，是一种细胞保护剂，可望用于临床，控制缺血再灌流损伤。