Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques (1) to identify possible sites of synthesis; (2) to compare the localization of ANP to known physiological effects; (3) to determine species differences, if any, in ANP localization; and (4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Apoptin Induces Chromatin Condensation in Normal Cells

Apoptin, a chicken anemia virus protein, was reported to induce tumor specific apoptosis, which was correlated with the nuclear localization of the protein in tumor cells. While in normal human cells, Apoptin was detected mainly in the cytoplasm and did not induce apoptosis. Using a recombinant adenovirus expressing Apoptin, we have found that Apoptin induced G₂-M cell cycle arrest and chromatin condensation in cancer cells. Here we report that adenovirus mediated Apoptin expression also induces G₂-M arrest in normal cells. In normal cells Apoptin is localized mainly in the cytoplasm but is also found in the nucleus of a subset of cells. Apoptin induces chromatin condensation not only when it is expressed in the nucleus but also when it is expressed in the cytoplasm. Our results indicate that Apoptin-induced chromatin condensation in the normal cells may not correlate with its nuclear localization and the mechanism of regulating the G₂-M transition might be a target for Apoptin.     

脑红蛋白在成年西门塔尔牛几种主要器官组织中的分布

目的探寻脑红蛋白(NGB)在成年西门塔尔牛不同组织细胞的分布特征。方法选择健康成年西门塔尔牛5只,利用免疫组织化学染色SP法观察NGB在其不同组织细胞的分布特征。结果结果显示,NGB在成年西门塔尔牛大脑和小脑皮质部、嗅球,海马,脊髓、肾上腺皮质部与髓质部、垂体、松果体、胰、睾丸、附睾、卵巢、子宫、胃、肠、肝、颌下腺、腮腺、心肌、脾脏、肺脏和肾脏等组织细胞中均有表达,NGB免疫阳性物质主要定位于细胞质。此外,本研究发现NGB在西门塔尔牛十二指肠段小肠腺细胞的表达强度要高于十二指肠腺细胞。结论NGB在成年西门塔尔牛多种器官组织细胞中的广泛分布,提示NGB不仅在氧的利用及功能活动中发挥作用,在其它生理活动中可能也担负着重要的功能。     

Immunohistochemical localization of metallothionein in human thyroid tumors.

High levels of metallothionein (MT) are present in the developing mammalian liver; however, a remarkable decrease is observed during postnatal life after weaning. This developmental profile is similar to that of certain oncofetal gene products such as alpha-fetoprotein, which is used as a tumor marker. This study deals with the reexpression of MT genes in thyroid tumors. With an immunohistochemical method, the presence of MT was investigated in tissue sections of normal and neoplastic human thyroid glands. Tissue sections of 34 thyroid tumors and 10 normal human thyroid glands were studied by means of the peroxidase-antiperoxidase method. MT was localized in 31 of the thyroid gland tumors. MT was also present in two of the normal thyroid glands. These findings indicate that although high levels of MT are mainly found in the fetal liver, it may also be expressed actively in certain human thyroid neoplastic tissues, and occasionally in normal thyroid tissue.     

The localization of delta antigen in the hepatocytes in chronic delta hepatitis

The results of immunohistochemical and electron microscopic examinations of liver biopsies from 47 patients with chronic delta hepatitis are presented. Delta antigen (HDAg) was detected by immunoperoxidase method in the liver of 35 (74.4%) patients. HDAg was demonstrated in hepatocyte nucleus alone in 22 (62.9%) patients, simultaneously in the nucleus and the cytoplasm in 9 (25.7%), in the cytoplasm alone in 4 (11.4%). In nuclear localization, delta antigen may fill the entire nucleoplasm or only the peripheral zone of the nucleus. When delta antigen fills the entire nucleus, hepatocytes undergo more marked pathological changes than those in which HDAg is localized in the nuclear periphery or those where no delta antigen was found.     

The proliferative status of haematopoietic progenitor cells in the developing murine liver and adult bone marrow

A class of primitive progenitor cells with high proliferative potential in vitro (HPP-CFC), has been identified in fetal liver and adult bone marrow both in murine and human systems. The kinetic properties of HPP-CFC₂ and the more mature granulocyte-macrophage colony forming units (CFU-GM) derived from murine fetal liver on d13, d15 and d19 of gestation, newborn liver and neonatal liver on d3 and d8 postpartum have been evaluated and compared with the kinetic properties of these progenitor cell populations derived from adult bone marrow. The frequency of HPP-CFC₂ in fetal liver was found to be greatest on d15 of gestation then subsequently declined in newborn and neonatal liver. Similarly, the highest proportion of HPP-CFC₂ engaged in DNA synthesis (53±3%) was detected in d15 fetal liver. This proportion decreased to 13±2% in the liver 1 wk after birth, which is comparable to the number of HPP-CFC₂ derived from adult BM which were in S-phase (10±1%). Production of CFU-GM was found to be greater in adult bone marrow than in either fetal or newborn liver. While the proportion of CFU-GM in S-phase was high in all 3 tissue samples, the greatest proportion of cycling CFU-GM (50±2%) was detected in d15 fetal liver. These results suggest that HPP-CFC₂ derived from fetal liver are actively cycling while HPP-CFC₂ derived from adult bone marrow are relatively quiescent. In contrast, a high proportion of CFU-GM derived from fetal, newborn liver and adult bone marrow are engaged in DNA synthesis.     

Immunochemical studies on the localization and on the concentration of cellular retinol-binding protein in rat liver during perinatal development

Studies were conducted to characterize the localization and the concentration of cellular retinol-binding protein (CRBP) in rat liver during perinatal development. The studies employed both a specific immunohistochemical staining procedure and a sensitive and specific radioimmunoassay for CRBP. Dramatic changes were seen in both the levels and localization of CRBP. Marked increases in hepatic CRBP levels were seen during the last week of gestation and the first postnatal week, resulting in a quadrupling of CRBP levels in this 2-week interval. During the second postnatal week CRBP levels remained very high (approximately 110 to 120 micrograms/gm of wet weight, with peak values at about day 11 of age). During the third postnatal week, CRBP levels declined, followed by a further decline to adult levels (40 to 50 micrograms/ml) postweaning. A changing pattern of immunohistochemical localization of CRBP was seen that correlated with the changes in CRBP levels. In fetal livers at days 11 to 13 of gestation, CRBP was selectively localized in perisinusoidal cells that resembled stellate (fat-storing) cells in their location and shape but that lacked the lipid droplets usually seen in these cells in adult liver. During the final week of fetal development and the first postnatal week, a progressive increase in CRBP in parenchymal cells was seen. By the second postnatal week, very strong staining for CRBP was seen in all parenchymal cells. The staining of parenchymal cells for CRBP then declined, and by weaning (day 21) the pattern of localization of CRBP in liver resembled that seen in the adult, with low to medium staining seen in parenchymal cells and strongly intense staining in perisinusoidal stellate cells. Thus, the rise and then decline in intensity of CRBP staining in parenchymal cells paralleled the pattern of changes seen in CRBP levels during the perinatal period. In the placenta, CRBP was selectively localized in the trophoblast layer of the chorioallantoic placenta and in the entodermal layer of the yolk sac placenta. These findings suggest that either retinol is metabolized or plays a functional role at these placental sites.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

Immunohistochemical localization of metallothionein in human testicular embryonal carcinoma cells

Summary The presence of high levels of metallothionein (MT) in developing mammalian cells is well documented. It has been suggested that the developmental profile and gene expression of MT is similar to that of the so-called oncodevelopmental gene products such as a-fetoprotein. In this study tissue sections of nine human embryonal carcinomas of the testis were tested by means of the avidin-biotin peroxidase complex for the presence of MT. The antigen was localized in variable amounts in the cytoplasm and nucleus in tumour cells in all cases. There was evidence that immunoreactivity was related to the histological growth pattern of tumour cells. These findings suggest that MT may be considered an oncodevelopmental product which could be useful as a tumour marker. In addition, the histology of these tumours might predict MT expression; this may prove of value in testing the hypothesis of MT-related emergence of drug-resistant cell lines in the course of treatment of tumours with metal-containing chemotherapeutic agents.     

Immunohistochemical analysis of calcitonin and calcitonin gene-related peptide in human lung

Calcitonin and calcitonin gene-related peptide (CGRP) have been localized immunohistochemically in neuroendocrine cells of normal and diseased human lungs. Cells immunoreactive for calcitonin and CGRP first appeared in immature bronchi in the 27th gestational week. Thereafter, both peptides were found in the same bronchial neuroendocrine cells throughout fetal and neonatal life. In adult lungs with or without neuroendocrine cell hyperplasia, only calcitonin was present. In 17 of 18 (94%) pulmonary tumorlets, variable numbers of calcitonin-positive cells were identified. A few CGRP immunoreactive cells, as a subset of calcitonin-containing cells, were found in only three (17%) lesions. Of 37 bronchial carcinoids, calcitonin was detected in 14 and CGRP was detected in 16 (38% and 43%, respectively), and both peptides were predominantly localized in the same cells. Ten of 45 (22%) small cell lung carcinomas were calcitonin-immunoreactive. CGRP was noted in only one (2%) of these tumors, and both peptides coexisted in single cells. These findings indicate that the patterns of calcitonin/CGRP expression in hyperplastic bronchial neuroendocrine cells, pulmonary tumorlets, and, to some extent, small cell lung carcinomas are similar to those of normal adult lungs. On the other hand, calcitonin/CGRP expression in bronchial carcinoids is similar to that of late fetal and neonatal lungs.     

野生型和突变型GDF5蛋白表达及亚细胞定位的研究

目的研究人生长分化因子5基因(GDF5)c.1118T>G(p.L373R)突变对GDF5蛋白在HEK-293细胞系表达及其亚细胞定位的影响。方法构建pDsRed1-N1-GDF5-WT和pDsRed1-N1-GDF5-L373R载体,分别转染HEK-293细胞,细胞培养72h后通过荧光显微镜观察比较野生型和突变型GDF5蛋白荧光分布情况。结果 GDF5蛋白在HEK-293细胞中成功表达,在荧光显微镜下观察到转染野生型和突变型GDF5的细胞胞浆均匀发出红色荧光,而空载体组的红色荧光则弥散于全细胞,荧光强度均匀一致。结论野生型和突变型GDF5蛋白均定位于HEK-293细胞质。     

Copine-3-增强型绿色荧光蛋白融合蛋白在细胞中的表达和定位

目的 构建增强型绿色荧光蛋白(pEGFP)-N1-CPNE3真核表达载体,检测Copine-3蛋白在细胞中的表达和定位.方法 利用RT-PCR从人支气管上皮细胞(HBE)中扩增得到编码Copine-3蛋白的CPNE3基因,并亚克隆至pEGFP-N1真核表达载体.将酶切鉴定和测序正确的pEGFP-N1-CPNE3重组质粒...     

Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung

The newborn lung is cleared of fetal liquid by active Na+ transport. The heterotrimeric (alpha, beta, gamma) epithelial Na+ channel, ENaC, mediates this process. To understand the role of individual ENaC subunits in Na+ transport during development, we quantified murine ENaC (mENaC) subunit messenger RNA (mRNA) expression levels of fetal, neonatal, and adult mouse lung by Northern blot analysis and studied regional expression by in situ hybridization. alphamENaC and gammamENaC mRNA expression increased sharply in late fetal gestation and reached near-adult levels by Day 1 of postnatal life. betamENaC expression increased more gradually through late fetal and early postnatal life and increased progressively until adulthood. In situ hybridization studies showed similar localization patterns of alphamENaC and gammamENaC subunit expression in fetal and postnatal lung. gammamENaC and alphamENaC subunits were initially localized to fetal lung bud tubules and by late gestation both subunits were expressed in all regions (acinar and bronchiolar) of the distal lung epithelium. betamENaC was detected from 16 d gestation onward and was expressed most intensely in small airways. There was little expression of betamENaC in the alveolar region. In postnatal lung all three subunits were expressed intensely in small airways. In adult lung, alphamENaC and gammamENaC were expressed in a pattern consistent with an alveolar type II (ATII) cell distribution. The timing of quantitative changes in mENaC subunit expression is consistent with a role of Na+ transport in liquid clearance of the perinatal lung. Intense expression of mENaC subunits in medium and small airway epithelium and in ATII cells suggests that these regions are a primary location for liquid absorption in the perinatal and postnatal murine lung.     

Cellular fibronectins are differentially expressed in human fetal and adult kidney

The localization of cellular forms of fibronectin (cFn) was studied in fetal and adult kidneys. We used monoclonal antibodies reacting with the extradomains A and B in cFN (EDAcFn, EDBcFn) as well as with a differentially glycosylated fetal form of the protein (Onc-cFn). In adult human kidney EDAcFn was present in glomerular mesangium and in the walls of larger blood vessels, whereas a polyclonal rabbit fibronectin antiserum widely reacted also with interstitial areas. Immunoreactivity for EDBcFn and Onc-cFn, however, was not found in adult kidney. In the basement membranes and interstitial areas of developing tubules and glomeruli the immunoreactivity for EDAcFn was distinct and detectable in the earlier stages also for EDBcFn. In developing glomeruli, EDAcFn and EDBcFn were detected in teh mesangial areas, but in more mature fetal glomeruli, the mesangial immunoreactivity only persisted for EDAcFn. Both EDAcFn and EDBcFn were found in the basement membranes in the medullary area of all developing kidneys. In fetal kidney, immunoreactivity for EDAcFn and EDBcFn was seen also in small blood vessels, including the capillaries. Immunoreactivity for Onc-cFn was found in mesangial cells of fetal glomeruli as well as in the intima of larger blood vessels but not in the basement membranes. The results show that the three forms of cFn are present in the fetal kidney and have certain differences in their distribution. Conversely, only the EDAcFn was detected in the adult kidney. The different, partially age-related distributions of the three types of cFns suggest that they may also differ in their functions.     

肿瘤转移抑制相关基因TMSG-1核仁定位信号序列的鉴定

目的 鉴定肿瘤转移抑制相关基因TMSG-1蛋白中潜在的特异性定位信号序列并探索其亚细胞定位机制.方法 聚合酶链反应(PCR)扩增TMSG-1开放读码框全长及不同长度的截断片段,定向克隆于绿色荧光蛋白(GFP)表达质粒pEGFP-N1;各融合蛋白表达质粒转染人胚肾细胞系HEK293细胞;转染48 h后提取细胞总蛋白进行GFP的Western blot检测或用冷丙酮固定细胞后激光共聚焦显微镜观察融合蛋白的亚细胞定位.结果 GFP分别融合TMSG-1全长蛋白(aa1-380)及其截断片段T1(aa1-70)、T2(aa1-128)、T3(aa129-380)、T4(aa71-128)、T5(aa71-179)和T6(aa71-380),Western blot检测结果显示成功表达了各融合蛋白.激光共聚焦显微镜观察亚细胞定位显示融合蛋白T4(aa71-128)主要定位于细胞核内的核仁部位,融合蛋白T6(aa71-380)以细胞核内弥散分布为主,而TMSG-1全长融合蛋白及融合蛋白T1、T2、T3、T5则定位于胞质.进一步的序列缺失去除T4( aa71-128)羧基末端10个氨基酸得到截断片段T4Δ119-128,T4Δ119-128融合的GFP仍位于细胞核,但核仁内的绿色荧光信号明显减弱.结论 肿瘤转移抑制相关基因TMSG-1存在潜在的核仁定位信号,位于aa119-128(RRRRNQDRPS),这一发现为深入研究TMSG-1的亚细胞定位及相关功能奠定了基础.