A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.     

PPAR-α Expression Inversely Correlates with Inflammatory Cytokines IL-1β and TNF-α in Aging Rats

Dehydroepiandrosterone (DHEAS) was given the name “fountain of youth” in reference to its beneficial properties in memory, cognition and aging. Cultured cell studies showed that DHEAS may mediate its action by counteracting aging-associated inflammation via PPAR-α activation. In the present study, we demonstrated an age-dependent increase in IL-1β and TNF-α expression in the brain and the spleen of aging rats, while PPAR-α expression was decreased in the spleen of 18 month-old rats. Oral treatment with DHEAS increased PPAR-α mRNA in 3 month-old rats and decreased PPAR-α protein expression in 18 month-old rats in the spleen. In contrast, DHEAS did not alter cytokine expression in spleen and brain of the three age groups. These findings underline a differential role for DHEAS in PPAR-α expression that is age-dependent, and also, that beneficial effects of DHEAS on cognitive function are unlikely mediated by a decrease in cytokine expression.     

Expression and localization of Parkinson's disease-associated leucine-rich repeat kinase 2 in the mouse brain

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinson's disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2-deficient mice is unaltered relative to wild-type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.     

Apoptosis of peripheral blood lymphocytes in patients with LRRK2-assoctated Parkinson's disease

Mutations in the Leucine Reach Repeat Kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson's disease (PD). Although the precise physiological and pathological role of LRRK2 is unclear, a direct link between mutant LRRK2 and apoptosis has been suggested. Using flow cytometric analysis (PI+Annexin V(FITC)) we showed increased spontaneous apoptosis of peripheral blood lymphocytes in patients with LRRK.2-associated PD compared to controls after 24 (P < 0.016) and 48 (P < 0.031 ) h of incubation (5 % CO2, 37 degrees C). We found the increased FAS mRNA level in peripheral blood lymphocytes of patients with LRRK2-associated PD compared to controls (P < 0.05) and to sporadic PD (sPD) (P < 0.002). Significant difference in FAS expression between patients with LRRK2-associated PD and controls remained after three years and was detected after 1 and 24 h during lymphocyte incubation (P < 0.03 and 0.05, respectively). Increased spontaneous lymphocytes apoptosis along to increased FAS expression in patients with LRRK2-associated PD suggest that LRRK2 mutations may lead to the activation of extrinsic apoptotic way.     

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.     

Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.     

Characterization of TAE684 as a potent LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.     

Different coexpressions of arthritis-relevant genes between different body organs and different brain regions in the normal mouse population

Structural changes in different parts of the brain in rheumatoid arthritis (RA) patients have been reported. RA is not regarded as a brain disease. Body organs such as spleen and lung produce RA-relevant genes. We hypothesized that the structural changes in the brain are caused by changes of gene expression in body organs. Changes in different parts of the brain may be affected by altered gene expressions in different body organs. This study explored whether an association between gene expressions of an organ or a body part varies in different brain structures. By examining the association of the 10 most altered genes from a mouse model of spontaneous arthritis in a normal mouse population, we found two groups of gene expression patterns between five brain structures and spleen. The correlation patterns between the prefrontal cortex, nucleus accumbens, and spleen were similar, while the associations between the other three parts of the brain and spleen showed a different pattern. Among overall patterns of the associations between body organs and brain structures, spleen and lung had a similar pattern, and patterns for kidney and liver were similar. Analysis of the five additional known arthritis-relevant genes produced similar results. Analysis of 10 nonrelevant-arthritis genes did not result in a strong association of gene expression or clearly segregated patterns. Our data suggest that abnormal gene expressions in different diseased body organs may influence structural changes in different brain parts.     

Distribution of PINK1 and LRRK2 in rat and mouse brain

Mutations in two kinases, PTEN induced kinase 1 (PINK1) and leucine-rich repeat kinase 2 (LRRK2), have been shown to segregate with familial forms of Parkinson's disease. Although these two genes are expected to be involved in molecular mechanisms relevant to Parkinson's disease, their precise anatomical localization in mammalian brain is unknown. We have mapped the expression of PINK1 and LRRK2 mRNA in the rat and mouse brain via in situ hybridization histochemistry using riboprobes. We found that both genes are broadly expressed throughout the brain with similar neuroanatomical distribution in mouse compared to rat. PINK1 mRNA abundance was rather uniform throughout the different brain regions with expression in cortex, striatum, thalamus, brainstem and cerebellum. LRRK2, on the other hand, showed strong regional differences in expression levels with highest levels seen in the striatum, cortex and hippocampus. Weak LRRK2 expression was seen in the hypothalamus, olfactory bulb and substantia nigra. We confirmed these distributions for both genes using quantitative RT-PCR and for LRRK2 by western immunoblot. As their broad expression patterns contrast with localized neuropathology in Parkinson's disease, the pathogenicity of clinical mutant forms of PINK1 and LRRK2 may be mediated by nigrostriatal-specific mechanisms.     

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.     

肝细胞生长因子mRNA在成年小鼠和人胎儿不同器官中的表达

本实验从成年小鼠和胎龄４－５月的人胎儿不同器官中分离总ＲＮＡ。经斑点印迹分析显示,肝细胞生长因子（ＨＧＦ）ｍＲＮＡ在成年ＫＭ小鼠多种器官中表达,其表达水平由高到低依次为：肺、肝、肾、卵巢、睾丸、大脑和胃;在脾、心、骨髓、小肠和骨骼肌组织中以ＨＧＦｍＲＮＡ。在胎龄４－５月的人胎儿中,ＨＧＦｍＲＮＡ表达水平由高到低依次为：大脑、肝、腮腺、胃、小肠、肾、心和骨骼肌;肺和脾组织为阴性。由此可见,ＨＧＦ在成     

Differential expression of rat beta-defensins

The innate immunity, utilizes a battery of broad-spectrum antibacterial cationic polypeptides (3-5 kDa) named alpha- and beta-defensins. Several beta-defensins have been isolated and shown to play a role in the defense of various tissues. Herein, we report the expression pattern of two rat beta-defensins, rBD-1 and rBD-2, in liver, kidney, lung, spleen, and brain using RT-PCR. To study polymorphism and verify gene identity, all cDNA products were sequenced. rBD-1 was expressed in the kidney, lung, brain, but not in spleen or liver, whereas rBD-2 was expressed in the lung, but not in the kidney or spleen. In addition, rBD-2 was expressed in the brain and liver. No polymorphism was found in the genes encoding rat beta-defensins. These findings demonstrate a different expression pattern for rBD-2 than what has been reported. We conclude that the rat may be a useful model to investigate the function and contribution of beta-defensins to host defense.     

GSK2578215A; A potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson's disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.     

脑红蛋白在赛加羚羊主要内脏器官中的表达与定位

赛加羚羊(Saiga tatarica)属于我国一级重点保护野生动物,其原产地主要为高寒低氧地区,现存种群则主要栖息于中亚地区的荒漠及半荒漠草原上。脑红蛋白是一种存在于脊椎动物体内具有运输和储存血氧能力的球蛋白,在动物适应低氧过程中具有重要的生理功能。为了初步探究赛加羚羊对低氧环境的耐受性机制,运用免疫组织化学染色法与实时荧光定量PCR技术,对脑红蛋白及脑红蛋白基因(NGB)在赛加羚羊的心、肝、脾、肺、肾等5种主要内脏器官中的分布规律与表达情况进行了探究。免疫组织化学染色结果显示,脑红蛋白在赛加羚羊的心、肝、脾、肺、肾中均有分布,阳性表达主要分布在其心肌细胞、肝细胞、脾白髓区中的淋巴细胞、肺泡细胞以及肾小球内皮细胞。实时荧光定量PCR结果显示,脑红蛋白基因在赛加羚羊心、肝、脾、肺、肾中的表达量不同,脾的表达量最高,心的表达量次之,两者均显著高于肝、肺和肾(P < 0.05);其后依次为肝、肾、肺,其中,肝的表达量显著高于肾和肺(P < 0.05),肾和肺之间表达量差异不显著(P > 0.05),肺的表达量最低。上述研究表明,脑红蛋白在赛加羚羊的主要内脏器官中均有阳性表达,不同内脏器官中的表达量不同,这表明脑红蛋白可能参与了这些内脏器官的氧利用过程,具体机制有待进一步探讨。     

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.     

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.