Identification of two insulin-like growth factor IIs in the Japanese eel, Anguilla japonica: cloning, tissue distribution, and expression after growth hormone treatment and seawater acclimation

To better understand the role of IGFs in Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-II (IGF-II) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-II cDNAs, eIGF-II-1 and eIGF-II-2, were cloned from the liver. A signal peptide and a mature peptide of both preproIGF-IIs were composed of 47 amino acids (aa) and 69 aa, but they differed at 17 aa and 13 aa, respectively. The E domain of eIGF-II-1 was 49 aa longer than that of eIGF-II-2, and differed at 22 aa within 52 aa. The highest eIGF-II-1 and II-2 mRNA levels were observed in the liver, with detectable levels also found in all tissues examined. The eIGF-II-1 mRNA levels in the liver, heart, and muscle were higher in females than in males, whereas those in the stomach and intestine were lower in the females. The eIGF-II-2 mRNA levels in the liver and swim-bladder were also higher in females than in males whereas those in the stomach, spleen, and intestine were lower in the females. The eIGF-II-1 mRNA levels in the liver were higher in large compared to small glass eels, while the eIGF-II-2 mRNA levels did not correlate with body weight. Both eIGF-II mRNA levels in the liver increased after eel GH treatments in vivo and in vitro. No differences in both eIGF-II mRNA levels were observed in the gills, liver, stomach and whole kidney between seawater- and freshwater-reared eels.     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

不同日龄大白猪视黄酸受体α基因组织表达

研究采用RT-PCR方法对大白猪的视黄酸受体α基因在1日龄、90日龄、180日龄、270日龄和360日龄的心、肝、胃、脾、肾、肺、大肠、小肠、肌肉、子宫、卵巢共11个组织的表达情况进行了研究。结果表明,RARαmRNA在肝、脾、肾、大肠、小肠、子宫和卵巢中持续表达,其中脾、大肠和小肠是持续高表达;180日龄时,所有组织的RARαmRNA的表达量普遍降低;360日龄时,所检的11个组织均高水平表达该基因。     

Localization of a putative cell adhesion molecule (gp110) in Wistar and Fischer rat tissues

A plasma membrane glycoprotein (gp110) involved in cellular adhesion was studied in Wistar and Fischer rats. For quantitative analysis of the gp110 molecule a sandwich-ELISA was used. High quantities of gp110 were found especially in the liver, small intestine, submandibular gland and lung. The distribution and localization of the gp110 were investigated by immunohistochemistry utilizing soluble complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase antibodies. Immunoreactivity was present in plasma membranes of vascular endothelial cells of some organs. Furthermore, immunostaining also occurred in plasma membranes of lymphocytes, exocrine gland cells, excretory duct cells, hepatocytes, epithelial cells of the small intestine, kidney and vesicular gland and in the cytoplasm of renal connecting and collecting duct cells. The localization of gp110 in the luminal domain of the plasma membrane at many sites suggests that this glycoprotein is also involved in processes distinct from cell adhesion.     

Tissue distribution of the 1,25-dihydroxyvitamin D3 receptor in the male rat.

Tissue distribution of 1,25-dihydroxyvitamin D3 receptors was studied in male rats using a quantitative immunoradiometric assay. Extracts were prepared from 16 different rat tissues and assayed for 1,25-dihydroxyvitamin D3 receptor. Measurable levels of receptor were detected in intestine, stomach, kidney, bone thyroid/parathyroid, skin, liver, spleen, heart and lung. The highest levels were found in the proximal small intestine and colon, containing over 1000 fmol/mg total protein, while ileum and kidney contained one-half and one-fourth of this amount, respectively. Other parts of the vitamin D endocrine system, including bone, thyroid/parathyroid and skin, contained moderate levels of receptor of 40 to 80 fmol/mg, while lung, heart, stomach, spleen and liver had levels at or below 20 fmol/mg. No 1,25-dihydroxyvitamin D3 receptor was detected in cerebrum, cerebellum or skeletal muscle. The data support a wide-spread role for 1,25-dihydroxyvitamin D3 on cellular processes and suggest a more important role for vitamin D in colon.     

Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues

We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.     

Specificity and distribution of mammalian carnosinase.

Hog kidney carnosinase (EC 3.4.13.3) was found to have a narrow specificity; it hydrolyzed carnosine, anserine and glycyl-L-histidine, but did not split L-alanyl-L-histidine or homocarnosine. The isoelectric point of this enzyme was 5.8 and its molecular weight was about 84 000. Carnosinase was found to be widely distributed in various tissues of the rat. Uterus, kidney, liver and lung contained high levels of carnosinase, whereas moderate concentrations were found in spleen, heart and brain, with low levels in small intestine, skeletal muscle and stomach, and none in blood.     

Localization of a putative cell adhesion molecule (gp110) in Wistar and Fischer rat tissues

Summary A plasma membrane glycoprotein (gp110) involved in cellular adhesion was studied in Wistar and Fischer rats. For quantitative analysis of the gp110 molecule a sandwich-ELISA was used. High quantities of gp110 were found especially in the liver, small intestine, submandibular gland and lung. The distribution and localization of the gp110 were investigated by immunohistochemistry utilizing soluble complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase antibodies. Immuno-reactivity was present in plasma membranes of vascular endothelial cells of some organs. Furthermore, immuno-staining also occurred in plasma membranes of lymphocytes, exocrine gland cells, excretory duct cells, hepatocytes, epithelial cells of the small intestine, kidney and vesicular gland and in the cytoplasm of renal connecting and collecting duct cells. The localization of gp110 in the luminal domain of the plasma membrane at many sites suggests that this glycoprotein is also involved in processes distinct from cell adhesion.Supported by the Deutsche Forschungsgemeinschaft (Re 523/3-3; Sfb 174) and the Fonds der Chemischen Industrie     

Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA

Three cDNA clones encoding rat basic fibroblast growth factor (FGF) were isolated from 10(6) independent clones prepared from a pregnant mare serum gonadotropin (PMSG)-stimulated rat ovarian cDNA library. One of the cDNA clones contained the entire coding sequence for basic FGF. The other two possessed the sequence coding the carboxy terminal 61 amino acids of rat basic FGF, the putative upstream intron sequence, and a 3'-noncoding region. The cDNAs encoding rat basic FGF predict a molecule consisting of 154 amino acid residues, which is one amino acid shorter than the human and bovine basic FGF. Otherwise, there are only 5 conservative amino acid substitutions between the rat and the human/bovine sequences. Poly A+ RNA from brain cortex and hypothalamus show a single 6.0 kb band that hybridizes to the cloned cDNA probe by Northern analyses. The observation that basic FGF mRNA is below the limits of detection in adrenal, spleen, heart, lung, kidney, liver, stomach, small intestine, large intestine, testis, and ovary support the notion that the that the high levels of the protein found in these tissues is due to storage of the mitogen in the extracellular matrix and not continuous gene expression. The significance of the abundance of mRNA in tissues which are not undergoing either active angiogenesis or cell proliferation (hypothalamus and brain cortex) is unclear but emphasizes the potential neuronotrophic function of basic FGF.     

版纳微型猪近交系 GHR 基因克隆及生物信息学分析简

目的 获得版纳微型猪近交系（BMI）生长激素受体基因（GHR）序列,通过生物信息学分析预测GHR功能并进行GHR mRNA多组织表达谱分析.方法 以版纳微型猪近交系的肝脏组织为材料提取RNA,RT-PCR方法扩增GHR基因编码区序列,将序列连接至pMD18-T载体进行克隆、测序和生物信息学分析;半定量PCR检测GHR mRNA在BMI不同组织中表达量的差异.结果克隆出了BMI GHR 编码区序列,提交GenBank获得登录号KC999114.该基因CDS长1917 bp,编码638个氨基酸.生物信息学分析表明,与长白猪的GHR序列相比BMI存在4处氨基酸替换,分别为p.E381D、p.A409S、p.L556V和p.A580G,均发生在胞内域.GHR基因多组织表达谱分析显示：GHR mRNA几乎在各组织中均有表达,在肌肉中表达量最高,在小肠、心、肝、神经纤维、脾、卵巢中表达量较高,在肺、胃、大脑、胰和肾中的表达量较低.结论 成功克隆了版纳微型猪近交系GHR全长编码区序列,进行了生物信息学功能分析和组织表达谱分析,为进一步阐明版纳微型猪近交系生长矮小机理奠定了基础.     

猪精氨酸-丝氨酸蛋白激酸3(SRPK3)基因的克隆、表达及多态性分析

通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3 (serine/arginine-rich specific kinase 3,SRPK3) 的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR (Real-time) 方法检测了SRPK3在1日龄和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS (1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A⁶²⁹→G⁶²⁹,T⁶⁵³→T⁶⁵³的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G¹⁰⁵⁹→ A¹⁰⁵⁹,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。     

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.