Expression of deafness protein Tmie in postnatal developmental stages of C57BL/6J mice

Loss-of function mutations in the transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). Previous studies report that the circling mouse can be an animal model for DFNB6. However, the expression pattern of Tmie protein in postnatal developmental stages has not been clearly revealed. In this study we tried to investigate the expression of Tmie protein in the liver, spleen, kidney, and lung, as well as in the cochlea. We examined various tissue samples from five different age groups of C57BL/6J animals. Using western blotting analysis, the expression of Tmie protein in these organs has been identified. The results show that Tmie protein expression in the cochlea has been increased in postnatal developmental stages, indicating that Tmie plays an important role in not only the development and also in the function of the cochlea. The expression pattern of Tmie in adult mouse organs such as the liver, spleen, kidney, and spleen significantly vary in adult rats. The order of Tmie expression level in mice (63 days after birth) was spleen, liver, lung, cochlea, and kidney, whereas in the adult rat it was liver, cochlea, lung, spleen, and kidney.     

小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。     

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.     

Expression level of Ubc9 protein in rat tissues

Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.     

分泌性中耳炎大鼠咽鼓管鼓室β-防御素的表达

目的检测大鼠β防御素(ratβ-defensin,rBD)在分泌性中耳炎大鼠咽鼓管鼓室的表达,探讨β防御素在分泌性中耳炎发病机制中的作用。方法排除中耳感染的清洁级SD大鼠48只,随机分为4组,前3组36只行颈部切口经右侧听泡注入脂多糖(lipopolysaccharide,LPS)溶液(1mg/mL)30μL制作分泌性中耳炎动物模型,造模后分别于第1、3、7天断头取咽鼓管鼓室黏膜;对照组12只右侧听泡注入生理盐水30μL,左侧听泡作为正常组,3d后断头取咽鼓管鼓室黏膜。逆转录聚合酶链反应(RT-PCR)检测咽鼓管鼓室rBD-1mRNA和rBD-2mRNA的表达。结果正常大鼠咽鼓管鼓室存在rBD-1和rBD-2的表达,且rBD-1的表达较rBD-2强,差异有统计学意义;造模后第1、3、7天,rBD-1表达变化不明显;rBD-2则在造模后第1、3天明显增加,差异有统计学意义,第7天渐回复到正常水平。结论在大鼠,rBD-1可能参与正常咽鼓管鼓室的防御功能,造模后rBD-2的表达增加可能与病原体入侵后的清除相关。     

Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

Tissue-specific expression of the rat glutathione S-transferases

Tissue-specific patterns of rat glutathione S-transferase expression have been demonstrated by in vitro translation of purified poly(A) RNAs and by protein purification. Poly(A) RNAs from six rat tissues including heart, kidney, liver, lung, spleen, and testis were used to program in vitro translation with the rabbit reticulocyte lysate system and [35S]methionine. The glutathione S-transferase subunits synthesized in vitro were purified from the translation products by affinity chromatography on S-hexylglutathione-linked Sepharose 6B columns. The affinity bound fractions were analyzed by Na dodecyl SO4-polyacrylamide gel electrophoresis and fluorography. A subunit of Mr = 22,000 detected in the in vitro translation products of poly(A) RNAs from heart, kidney, lung, spleen, and testis is missing from the translation products of liver poly(A) RNAs. This Mr = 22,000 subunit is present only in the anionic glutathione S-transferase fraction purified from rat heart, kidney, lung, spleen, and testis. Purified anionic glutathione S-transferase from rat liver does not contain this subunit. The relative specific activities toward a dozen different substrates also demonstrate the nonidentity between liver and kidney anionic glutathione S-transferases. In addition, among the glutathione S-transferase subunits expressed in the liver, some of them could not be detected in the other tissues investigated. Our results indicate that tissue-specific expression of rat glutathione S-transferases may occur pretranslationally.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy

Adrenomedullin (AM) is a potent vasorelaxing peptide originally isolated pheochromocytoma. Recently, a family of receptor-activity-modifying proteins (RAMPs 1-3) were identified in humans. Associated with the calcitonin receptor-like receptor (CRLR), RAMP2 or RAMP3 may function as the AM receptor. Here we cloned rat RAMP family, analyzed their distribution in rat tissues, and examined regulation of their expression in the kidney using an obstructive nephropathy model. Northern blot analyses revealed that the RAMP family genes are expressed in various tissues with different tissue specificity; RAMP1 is abundantly expressed in the brain, fat, thymus, and spleen, RAMP2 in the lung, spleen, fat, and aorta, while RAMP3 is most abundant in the kidney and lung. After ureteral obstruction, RAMP1, RAMP2, and CRLR gene expressions in the obstructed kidney were markedly upregulated, whereas RAMP3 expression was unchanged. Thus, RAMPs are regulated differently in obstructive nephropathy, suggesting their distinct roles in renal pathophysiology.     

Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues

(1) The tissue-specific expression of various glutathione-dependent enzymes, including glutathione S-transferase (GST), glutathione peroxidase and glyoxalase I, has been studied in bovine adrenals, brain, heart, kidney, liver, lung and spleen. Of the organs studied, liver was found to possess the greatest GST and glyoxalase I activity, and spleen the greatest glutathione peroxidase activity. The adrenals contained large amounts of these glutathione-dependent enzymes, but significant differences were observed between the cortex and medulla. (2) GST and glyoxalase I activity were isolated by S-hexylglutathione affinity chromatography. Glyoxalase I was found in all the organs examined, but GST exhibited marked tissue-specific expression. (3) The alpha, mu and pi classes of GST (i.e., those that comprise respectively Ya/Yc, Yb/Yn and Yf subunits) were all identified in bovine tissues. However, the Ya and Yc subunits of the alpha class GST were not co-ordinately regulated nor were the Yb and Yn subunits of the mu class GST. (4) Bovine Ya subunits (25.5-25.7 kDa) were detected in the adrenal, liver and kidney, but not in brain, heart, lung or spleen. The Yc subunit (26.4 kDa) was expressed in all those organs which expressed the Ya subunit, but was also found in lung. The mu class Yb (27.0 kDa) and Yn (26.1 kDa) subunits were present in all organs; however, brain, lung and spleen contained significantly more Yn than Yb type subunits. The pi class Yf subunit (24.8 kDa) was detected in large amounts in the adrenals, brain, heart, lung and spleen, but not in kidney or liver. (5) Gradient affinity elution of S-hexylglutathione-Sepharose showed that the bovine proteins that bind to this matrix elute in the order Ya/Yc, Yf, Yb/Yn and glyoxalase I. (6) In conclusion, the present investigation has shown that bovine GST are much more complex than previously supposed; Asaoka (J. Biochem. 95 (1984) 685-696) reported the purification of mu class GST but neither alpha nor pi class GST were isolated.     

The influence of chromium chloride consumption on lipid peroxidation and activity of the antioxidant defense in rat tissues

The effect of food supplementation with chromium (CrCl₃ · 6H₂O) on intensity of peroxide processes and activity of antioxidant enzymes has been investigated in some rat tissues. Food supplementation with 200 μg/kg CrCl₃ · 6H₂O for 30 days resulted in the increase of tissue chromium. The tissue chromium content of chromium-treated rats decreased in the following order: spleen, heart, kidney, lung, brain, liver, skeletal muscles. All organs and tissues (except skeletal muscles) of chromium-treated rats were characterized by decreased content of lipid peroxidation (LPO) products: hydroperoxides and thiobarbituric acid reactive substances (TBARS). The maximal reduction in LPO products was observed in spleen, kidney, liver, and lung. Treatment with chromium also caused an increase in the activity of glutathione peroxidase, glutathione reductase, and calatase in all tissues and organs studied. In the brain and kidney an increase in the content of reduced glutathione was observed. Superoxide dismutase activity was higher in myocardium and skeletal muscles, basically equal in lung and liver, while in other organs (brain, kidney, spleen) of experimental animals it was lower than in control animals. Results of this study suggest that chromium exhibits tissue/organ-specific regulatory effects on enzymes of the antioxidant defense     

High expression of the mRNA of cytochrome P450 and phase II enzymes in the lung and kidney tissues of cattle

The objective of this study was to investigate the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronsyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues (liver, mammary gland, lungs, spleen, kidney cortex, heart, masseter muscle and tongue) of cattle, using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined, including the liver, with the highest expression level in the kidney. CYP1A2-, 2E1- and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in the lung tissue, while CYP2C9-like mRNA was expressed in the liver and kidney tissues of the cattle examined. UGT1A1- and GSTA1-like mRNAs were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in the kidney. The high expression of UGT1A1 in the lung tissue and GSTA1 in the liver tissue was unique to cattle; this has not been reported for rats or mice. The findings of this study strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotics metabolism.     

Molecular cloning and characterization of a novel mouse betaPix isoform

BetaPix, a Pak-interacting guanine nucleotide exchange factor is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. Currently, three 1Pix isoforms, betaPix-a, -b, and -c have been reported. In this study, the cDNA of a novel Pix splice variant was isolated from a mouse brain cDNA library. The cloned betaPix isoform, named betaPix-d, lacks leucine zipper domain that is present in other Pix isoforms, and has a 11 amino acid addition at carboxyl terminus and distinct 3'-UTR Analysis of the tissue distribution of betaPix-d using RT-PCR revealed that its message was present mainly in brain and testis but in lower levels in heart, spleen, lung, liver, skeletal muscle and kidney. In situ hybridization studies with the 13Pix-d specific probes in the rat embryo show that betaPix-d isoform is expressed mainly in the central nervous system. Moreover, temporal expression pattern of the isoform is correlated with the active neurogenesis period in the cerebral cortex and cerebellum during rat brain development. These findings suggest that betaPix-d isoform may be developmentally regulated.     

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase-B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.