Hypoxia-induced upregulation of endothelial small G protein RhoA and Rho-kinase/ROCK2 inhibits eNOS expression

The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3h of hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of hypoxia, but was significantly decreased after 5h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.     

A comparative study on the synthesis of dopamine in the human,dog and rat kidney

The present work has examined the ability of human, canine and rat renal tissues to synthesize dopamine from added L-3,4-dihydroxyphenylalanine (l -DOPA); the deamination of newly-formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also studied. In some experiments, slices of renal cortex obtained from the human, dog and rat kidneys were used; tissues were incubated with increasing concentrations (5–5000 μM) of l -DOPA. The accumulation of newly-formed dopamine was, in all three species, found to be dependent on the concentration of l -DOPA, being the rat renal tissues endowed with a greater ability to produce dopamine, followed by the human and the dog tissues. In experiments performed in kidney homogenates, the decarboxylation of l -DOPA into dopamine was also found to be dependent, in all three species, on the concentration of l -DOPA used (10–5000 μm). AAAD activity as determined in kidney homogenates was found to be in the rat kidney (V_max = 7.7±0.8 nmol mg^-1 protein h^-1) higher than that occurring in the human (V_max = 5.8±0.6 nmol mg^-1 protein h^-1) and the dog kidney (V_max = 3.9 ± 0.5 nmol mg^-1 protein h^-1). No statistically significant differences were found between the K_m values of the three species (human, 62±8 μM; dog, 54±6 μM; rat, 82±12 μM). A considerable amount of newly-formed dopamine in both kidney slices and homogenates was converted into DOPAC; the DOPAC /dopamine ratios in these experimental conditions were greater in the human kidney, followed by the rat and dog. It is concluded that the decarboxylation of l -DOPA into dopamine is greater in the rat kidney followed by the human and dog, whereas the deamination of the amine into DOPAC appears to be greater in human renal tissues.     

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function.Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.     

Release of urodilatin from perfused rat kidney and from cultured neonatal rat kidney cells

Release of rat urodilatin (rURO) from isolated perfused rat kidneys and neonatal rat kidney cells could be demonstrated by a specific competitive radioimmunoassay (rURO-RIA) using [¹²⁵I]rURO as the competitive antigen and an antiserum against the hypothetical rURO-N-terminus, Ala-Gly-Pro-Arg, as concluded from the amino acid sequence of the rat prohormone CDD/ANP-1-126. This antiserum did not react with synthetic rCDD/ANP-99-126, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), or human URO (hURO). rURO could be demonstrated in the urine of the perfused rat kidney after an equilibration period of 20 min. After an initial slight decrease in the second 20 min, rURO production remained at almost the same level during the perfusion time of 100 min. A total of 470 fmol · 10 min^–1 · g^–1 kidney rURO was produced within 80 min. rURO was also produced by neonatal rat kidney cells kept in serum-free Dulbecco's modified Eagles medium. The production of rURO depended on the cultivation time of the cells. It increased up to 3 days reaching 239 ±7.5 fmol-h^–1 · g^–1 protein, afterwards it decreased rapidly. The results obtained indicate that the rat kidney produces a peptide of the type A family of natriuretic peptides, which very likely represents the putative rURO.     

人胆酸转运蛋白基因的克隆、亚细胞定位及其在肾组织中的表达

目的：克隆人肾与小肠ASBT（顶端Na+/胆汁酸协同转运蛋白）基因并比较2者的序列差别，明确ASBT蛋白在肾小管上皮的亚细胞定位及在人肾组织中的表达情况。方法:从人肾和小肠组织中提取总RNA，然后用带有8肽FLAG标签的PCR引物通过RT-PCR技术扩增ASBT全长cDNA基因并测序，并将其插入真核表达载体中构建ASBT蛋白真核表达载体，然后将其转染到肾小管上皮细胞LLC-PK1中表达并用免疫荧光-激光共聚焦显微镜观察该蛋白的亚细胞定位情况。用免疫组化技术观察ASBT在人肾组织中的表达分布。结果:序列分析结果表明肾小管ASBT基因的序列与小肠ASBT序列完全一致。Western blotting表明ASBT基因在LLC-PK1细胞中得到了正确的表达。共聚焦显微镜分析显示正常ASBT蛋白主要定位于肾小管上皮细胞膜上，与生物信息学的预测结果一致。免疫组化染色表明ASBT蛋白主要表达于人近端肾小管上皮的刷状缘侧，在间质及远端小管没有表达。结论:人肾小管ASBT基因序列与小肠ASBT相同，ASBT蛋白主要表达于近端肾小管上皮细胞管腔侧细胞膜。     

Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex

Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified anti-alpha-1 antibody was confirmed to react only with a single band of about 70-80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. Moreover, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons.     

Cellular and subcellular immunohistochemical localization of angiotensin-converting enzyme in the rat adrenal gland

Angiotensin-converting enzyme (ACE) was localized by immunocytochemical methods in the rat adrenal gland. The three-layer peroxidase-antiperoxidase method was used with light and electron microscopy. At the cortical level, ACE immunoreactivity occurred only on the luminal side of vascular endothelial cell membranes in the adrenal capsule, whereas no ACE activity was found in the zona glomerulosa, fasciculata, or reticularis. ACE immunoreactivity was found mainly but not exclusively on the luminal side of all the types of vessels in the adrenal medulla such as capillaries, venous sinuses, and arteriae medullae. In addition, ACE was localized on the plasma membrane of chromaffin cells but never in Schwann cells nor in nerve fibers. These results provide evidence for a local production of angiotensin II at the vascular level in the adrenal gland. In addition, the presence of ACE on the plasma membrane of chromaffin cells suggests that angiotensin II or other peptides, such as bradykinin, could be metabolized here.     

SIRT6在大鼠脑组织中的表达与分布

目的研究SITR6蛋白在正常大鼠脑组织中的表达与分布。方法在常温下自由饮水与饮食的情况下喂养8周的大鼠,采用免疫组织化学检测大鼠脑组织中SIRT6的表达与定位。结果 SIRT6是一种主要在神经元中表达的核蛋白,在大鼠正常脑组织中广泛表达,尤其在大脑组织中的皮质、髓质、海马中的神经元表达量较高;且在海马集中表达和在大脑皮质的外锥体细胞层和内椎体细胞层较明显,而在大脑髓质中表达低于海马和皮质。结论 SIRT6作为一种核蛋白,为研究SIRT6的功能提供了新的思路。     

Inhibitors caveolin-1 and protein kinase G show differential subcellular colocalization with Nitric oxide synthase

Background

Nitric oxide synthase (NOS) is negatively regulated by protein-protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells (SMC). The PKG is also found in endothelial cells and it inhibits activated NOS within endothelial cells.

Methods

We used confocal fluorescence microscopy to colocalize the inhibitors caveolin-1 and PKG with NOS in freshly isolated neonatal lamb endothelial cells in order to corroborate the speculation of their differential effects on NOS. The roles of caveolin-1 and PKG as regulators of NOS were investigated by examining their respective subcellular sites of colocalization with NOS using qualitative fluorescence immunohistochemistry and confocal microscopy.

Results

Caveolin-1 was colocalized with NOS in the plasma membrane and Golgi. The PKG1-beta isoform was colocalized with serine116 phosphorylated NOS in the cytosol and in vesicular structures seen in the endoplasmic reticulum and in the nuclear region.

Conclusion

We conclude that unlike caveolin-1, a known pre-activation inhibitor of nascent NOS, PKG may be a post-activation inhibitor of NOS, possibly important for the recycling of the spent enzyme.     

Expression of APOBEC3G in kidney cells

The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a member of the APOBEC family possessing DNA mutator activity through cytosine deamination, is reported to play an important role in host defense against infections such as those of hepatitis B virus and human immunodeficiency virus. Here, we examined the expression of APOBEC3G in human kidney cells to better understand its biological role against infection. APOBEC3G was immunohistochemically detectable in kidney mesangial cells and also to some extent in kidney epithelial tubular cells. In addition, overexpression of APOBEC3G was shown in renal carcinoma tissues and cell lines. APOBEC3G expression was upregulated by inflammatory cytokines, such as interferon, interleukin-6, and tumor necrosis factor. These results may provide new insight into the role of APOBEC3G in host defense against viral infection and cancer.     

T细胞衔接活化因子-绿色荧光蛋白真核表达质粒的构建及其在Jurkat细胞的定位

目的:构建增强型绿色荧光蛋白(EGFP)与T细胞衔接活化因子(Linker for activated ofT cells,LAT)融合蛋白的真核表达载体,观测LAT-EGFP在Jurkat细胞中的定位表达.方法:利用RT-PCR技术提取并扩增LAT除去终止密码子外的全部序列,克隆到真核表达载体PEGFP-N3,酶切鉴定并测序.瞬时转染到Jurkat细胞中进行表达,荧光共聚焦显微镜观察LAT-EGFP在Jurkat细胞中的表达及细胞定位.提取转染后细胞总RNA,通过RT-PCR的方法检测LAT-EGFP在转录水平的表达.利用Western blot法进一步鉴定融合蛋白的表达.结果:重组载体经酶切鉴定,切出片段长度在750 bp左右与插入序列长度相符,并进一步进行测序鉴定证实连接完全正确.共聚焦显微镜观察表达的LAT-EGFP融合蛋白定位在细胞膜上,并呈点簇状聚集状态.RT-PCR扩增证实了LAT和EGFP的融合蛋白在Jurkat细胞中在转录水平的表达,Western blot分析进一步证明了LAT和EGFP融合蛋白构建成功,并在蛋白水平上有明显的融合表达.结论:成功构建真核表达载体LAT-EGFP,且融合蛋白LAT-EGFP与野生型LAT在Jurkat细胞中的定位一致,具有功能表达效应,这为后续准确研究具有棕榈酰化位点的跨膜接头蛋白的信号转导作用提供了一种良好的研究载体和方法.     

Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.     

RhoA在内皮-单核细胞激活多肽-Ⅱ增强血肿瘤屏障通透性中的作用

目的探索信号分子RhoA在内皮-单核细胞激活多肽-Ⅱ(endothelial monocyte activating polypeptide-Ⅱ,EMAP-H)增强血肿瘤屏障(blood-tumor barrier,BTB)通透性中的作用。方法采集出生3~5d的Wistar胎鼠的大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)原代培养;将BMECs与C6脑胶质瘤细胞共培养,构建体外BTB模型;共培养后的BMECs随机分成3组(每组6例):对照组、EMAP-Ⅱ组和C3 exoenzyme+EMAP-Ⅱ组。测定跨内皮阻抗值和辣根过氧化物酶流量评估各组BTB通透性变化情况;Western blot法检测BMECs上紧密连接相关蛋白ZO-1的表达水平;免疫荧光法检测ZO-1和细胞骨架蛋白F-actin在BMECs上的分布与表达。结果与对照组相比较,EMAP-Ⅱ组BTB通透性显著增高,ZO-1和F-actin在BMECs上的表达水平显著降低,应力纤维形成明显增多;EMAP-Ⅱ的上述作用受到RhoA抑制剂C3 exoenzyme预处理的显著抑制。结论信号分子RhoA在EMAP-Ⅱ增强BTB通透性中发挥着重要的作用。     

Mutations of signal-transducing G proteins in human disease

Heterotrimeric guanine nucleotide binding proteins (G proteins) couple a large number of cell surface receptors to their intracellular effector molecules, such as enzymes or ion channels. Mutations of G proteins can lead to either activation or inactivation of the corresponding signal transduction pathway and thus cause clinical symptoms. Mutations of heterotrimeric G proteins have been found in a number of endocrine tumors, the McCune-Albright syndrome, Albright's hereditary osteodystrophy, and a combination of precocious puberty and pseudohypoparathyroidism Ia. The identification of the molecular defects underlying the above disorders and the investigation of their functional consequences for metabolism and growth regulation have been the subject of many studies over the past few years. A close understanding of these pathophysiologic mechanisms is crucial for the development of therapeutic strategies.Abbreviations AHO Albright's hereditary osteodystrophy - G protein Guanine nucleotide binding proteins - Gs protein Stimulatory G protein - Gi protein Inhibitory G protein - GHRH Growth hormone releasing hormone - MAS McCune-Albright syndrome - PHP Pseudohypoparathyroidism - PPHP Pseudopseudohypoparathyroidismus - PTH Parathormone     

Expression of the matrix metalloproteinases 2 and 9 in the rat small intestine during intrauterine and postnatal life

The expressions of matrix metalloproteinases 2 and 9 have been described during the development, as an example in heart and tooth but not in the small intestine yet. In this context, this study aimed to evaluate the expressions of MMP-2 and MMP-9 in the small intestine of Wistar rats during intrauterine (IU) and postnatal (PN) life. Expressions were determined on the 15th and 18th days of IU life and the 3rd, 10th, 17th, 25th, and 32nd days of PN life. Intestinal samples obtained from six animals were submitted to zymography, immunohistochemistry, and staining with Masson's trichrome. The results showed that MMP-2 and MMP-9 were not expressed during IU life; however, after birth, MMP-9 was immunolocalized in the goblet and mast cells. In conclusion, our results showed that MMP-2 and MMP-9 were not expressed in absorptive epithelial cells during the IU period of the small intestine but after birth, MMP-9 was expressed in the goblet cells, and mast cells present in the lamina propria.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

CD2-associated protein in human urogenital system and in adult kidney tumours

We studied expression of CD2-associated protein (CD2AP) in human urogenital system and in adult kidney tumours. In the cortex of normal kidney, CD2AP was expressed in all types of tubules and in the glomeruli. Labelling was more intense in cytokeratin 7- and in Tamm–Horsfall-positive tubules than in proximal tubules. In the medulla, expression was observed in the collecting ducts. Urothelium and the epithelium of prostatic acini, seminal vesicles, seminiferous tubules, epididymal ducts, Fallopian tube, endometrium and endocervix as well as granulosa cells showed moderate to strong CD2AP positivity. In syncytiotrophoblast, the expression was weaker than in cytotrophoblast. Endometrial stroma was negative, but decidualised stroma was weakly positive. Clear cell renal cell carcinoma (RCC) (n=63) showed a weak expression. Type-I papillary RCCs (n=4) and papillary adenomas (n=3) were negative. The epithelium lining the cysts in multilocular cystic RCCs (n=3) and in cystic nephroma (n=1) was strongly positive. Chromophobe RCCs (n=2), oncocytomas (n=3) and urothelial carcinomas (n=2) were moderately positive. The results show that CD2AP displays a specific expression pattern in human urogenital organs and that distinct expression is shown in several types of kidney tumours but not in type-I papillary RCCs or in papillary adenomas.     

Analysis of the human serological immune response to a variable region of the attachment (G) protein of respiratory syncytial virus during primary infection

The serum antibody responses of babies to the variable carboxy-terminal region of the attachment (G) protein of respiratory syncytial virus (RSV) have been analysed using paired acute and convalescent sera from infants experiencing their first RSV infection with viruses of known genotype. The variable 84–85 carboxy-terminal amino acids of the G protein of six recent isolates of group A RSV were expressed in Escherichia coli as fusion proteins with glutathione S-transferase. About half the infants developed antibodies which recognised these fusion proteins. The patterns of response obtained in enzyme linked immunosorbant assays and immunoblotting assays were closely related to the infecting genotype. © 1996 Wiley-Liss, Inc.