Calcitonin gene-related peptide is not essential for the development of pressure overload-induced hypertrophy in vivo.

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCbeta and skeletal alpha-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1-2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP.     

Agiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats

Recently, the calciumcalmodulindependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and MHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in Ang II treated rats, suggesting that the calciumcalmodulindependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.     

Calcitonin gene-related peptide (CALCA) is a proangiogenic growth factor in the human placental development

Recent studies have shown that homozygous knockout of gene for calcitonin gene-related peptide (CALCA) receptor component, calcitonin receptor-like receptor (CALCRL), led to extreme hydrops fetalis and embryonic death, underlining the critical role of CALCA in embryonic development and fetal growth. The present study was designed to determine the cellular localization of CALCA and its receptor components, CALCRL and receptor activity modifying protein 1 (RAMP1), at the human implantation site during early pregnancy; to assess whether CALCA regulates in vitro angiogenesis of human endothelial cells; and to examine whether CALCA can improve angiogenic imbalance in preeclamptic placental explants. Our studies demonstrated that both protein and mRNA for CALCA were expressed by the villous and extravillous trophoblasts and decidual cells in the first-trimester villous tissues. CALCA receptor components, CALCRL and RAMP1, were expressed by both villous and extravillous trophoblast cells, as well as vascular endothelial cells. CALCA induced both endothelial proliferation and migration in a dose- and time-dependent manner, and it promoted capillarylike tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel. CALCA-induced angiogenesis of human endothelial cells was completely blocked by CALCA antagonist CALCA(8-37). Further, conditioned medium from preeclamptic placental explants significantly inhibited HUVEC capillarylike tube formation compared with gestational age-matched controls, and conditioned medium from preeclamptic placental explants incubated with CALCA significantly improved capillarylike tube formation. We conclude that CALCA induces in vitro angiogenesis by stimulating endothelial cell proliferation, migration, and capillarylike tube formation; thus, CALCA at the human implantation site may constitute a potential autocrine or paracrine mechanism that could modify placental angiogenesis and neovascularization.     

Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.     

大鼠不同心肌肥厚模型左心室基因表达谱变化的比较

为了解心肌肥厚时基因表达谱的变化规律,本实验复制了三种大鼠心肌肥厚模型：肾上腹主动脉缩窄(suprarenal abdominal aortic stenosis,SRS)、动静脉瘘(arterial-vein fistula,AVF)和去甲。肾上腺素持续静脉输注(jugular vein infusion of norepinephrine,NEi),并应用组织化学方法和超声心动术检测大鼠心脏结构和功能指标,应用cDNA基因芯片技术检测心脏基因表达水平的变化。SRS和NEi引起大鼠向心性心肌肥厚,AVF引起大鼠离心性心肌肥厚,其中NEi大鼠心肌纤维化明显。对不同心肌肥厚模型间大鼠左心室基因表达谱的变化进行两两比较。结果显示,有部分基因在不同模型中表达水平均发生变化,其中多数基因在两种模型中表达水平改变的方向相同,也有少部分基因在两种模型中表达水平改变方向相反。综合比较三种心肌肥厚模型的基因表达谱,各种模型都有特异的基因表达变化,但是有19个基因在三种心肌肥厚模型中表达水平均发生改变。研究结果有可能成为心肌肥厚的标志性基因或治疗靶点,为心肌肥厚发生机制的深入研究提供了新的线索。     

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKC, PKC and PKC, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKC levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKC expression levels were also unaffected during the induction of LVH, but increased 3.2 ± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKC levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKC was not increased at any time point. In contrast, PKC autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKC autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKC, whereas total PKC levels were markedly increased (6.0 ± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKC levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKC may be involved in the induction of pressure overload LVH, whereas both PKC and PKC may be involved in the transition to heart failure.     

The glycine decarboxylase complex is not essential for the cyanobacterium Synechocystis sp. strain PCC 6803

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.     

降钙素基因相关肽转基因预防小鼠自身免疫性糖尿病发病及其抗氧化应激机制

有证据表明,活性氧(reactive oxygens pecies,ROS)参与了胰岛β细胞自身免疫损伤,是自身免疫性糖尿病发病的重要原因之一。一氧化氮(NO)和过氧化氢(H2O2)介导了致炎性细胞因子对胰岛B细胞的损害,引起脂质过氧化反应,进而损伤胰岛细胞。降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在心肌细胞中可抑制ROS生成而具有细胞保护作用。通过CGRP裸质粒肌肉注射体细胞电针强化转基因方法,使骨骼肌持续表达CGRP,观察其对小剂量多次注射链脲佐菌素(streptozotocin,STZ)造成的小鼠自身免疫性糖尿病发病的影响,进一步测定胰腺局部活性氧含量以及抗氧化酶的改变,探讨其抗氧化应激机制。结果发现,CGRP裸质粒直接注射入小鼠双后肢胫前肌。继以程控电针刺激导入(体内电穿孔法),可使血浆和骨骼肌组织CGRP表达水平显著增高,且持续4周以上;注射STZ同时给予CGRP转基因治疗可减轻胰岛β细胞损伤,显著降低自身免疫性糖尿病的发病率及血糖水平;CGRP转基因可显著抑制自身免疫性糖尿病小鼠胰腺局部活性氧和丙二醛的生成,增加过氧化氢酶(CAT)及超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果提示,CGRP裸质粒直接注射、电针辅助导人转基因可获得CGRF持续高水平的表达,能够预防小鼠自身免疫性糖尿病的发病,其机制之一可能为CGRP抑制了活性氧对胰岛β细胞的损伤。     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

MiR-143 is not essential for adipose development as revealed by in vivo antisense targeting

MiR-143 plays an important role in promoting the adipogenic differentiation of pre-adipocytes. Here, we report that systematic silencing of miR-143 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any obvious abnormalities in the adipose tissue differentiation. Furthermore, there were no significant differences in the expression level of several adipogenic marker genes, such as PPARγ and C/EBPα, in these animals compared with the controls. Therefore, we hypothesize that miR-143 may function as a fine tuning molecule rather than as a switch in the adipogenic regulatory network in mice. In addition, the proposed miR-143 target, ERK5, which was previously identified in human preadipocytes, was not effectively inhibited by miR-143 either in the murine preadipocyte cell line, 3T3-L1, or in primary mouse adipose tissue. However, we did fibroblast growth factor 7 (Fgf7) was identified as a target of miR-143 in murine adipogenesis.     

Calcitonin and calcitonin gene-related peptide alter the excitability of neurons in rat forebrain

Individual neurons in the hypothalamus, thalamus, cortex, and other forebrain areas of urethane-anesthetized, male rats were iontophoretically tested for their membrane sensitivity to salmon calcitonin (CT), human CT, and CT gene-related peptide (CGRP). Extracellular recording of unit activity revealed that depression of neuronal firing was the predominant effect of iontophoretically applied salmon CT (35 of 74 cells tested). Few neurons responded to salmon CT with an increase in firing rate (N = 3). When CGRP was iontophoretically applied a pattern of response resembling that of salmon CT was observed. CGRP was predominantly inhibitory and excited those neurons whose firing rate was increased by salmon CT. Inhibition was also the predominant effect of human CT. However, no neurons were excited by human CT. The results clearly demonstrate that a subpopulation of neurons with membrane sensitivity to salmon CT, human CT, and CGRP are present in the rat forebrain. This finding suggests that modulation of neuronal activity may underlie the behavioral and biochemical effects of these peptides when administered centrally. Endogenous CGRP and CT-like peptides in rat brain may be capable of regulating these events as neurotransmitters or neuromodulators.     

Studies on the Lipoprotein Lipases of Microorganisms

Mucor javanicus IAM 6108 was cultivated aerobically at large scale in the medium containing corn steep liquor 3.0%, soluble starch 1.0%, soybean yuto 1.0% and inorganic salts, and the lipoprotein lipase produced was recovered by addition of ammonium sulfate (0.7 saturation). From this crude preparation, the enzyme was purified about 13 times, through ammonium sulfate fractionation (0~0.4 saturation), precipitation at pH 4.0, ethanol precipitation (80%) and Sephadex G-200 gel filtration. The purified lipoprotein lipase was sedimented as single peak in ultracentrifugal analysis in the presence of 1.0% sodium dodecylsulfate. The enzymatic properties of the purified enzyme was as follows; optimum pH was 7.0, stable pH range was from 5.0 to 7.0, optimum temperature was 40°C, inactivated rapidly above 50°C. The lipoprotein lipase activity was inhibited by 75% and 88% by 10^?2 m taurocholate and 1.0 m NaCl, respectively. ZnCl₂, CuCl₂, Pb(NO₃)₂, and SnCl₂ at 10^?3 m showed complete inhibition. The ratio of lipoprotein lipase to lipase activity was 10 : 1. Lipoprotein lipase activity was dependent on the concentration of blood plasma which could be substituted by bovine serum albumin or egg albumin to a certain degree. The results suggesting the preferential α-fatty acid hydrolysis was obtained.     

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant

Summary Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n=23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1–3 months, n=5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.     

Calcitonin gene-related peptide increases the production of glycosaminoglycans but not of collagen type I and III in cultures of rat fat-storing cells

We investigated whether calcitonin gene-related peptide (CGRP) was able to affect the production of collagen and glycosaminoglycans (GAG) in cultures of rat fat-storing cells (FSC). Rat CGRP (1 nM-1 microM) induced a dose-dependent increase of total GAG production in FSC cultures with an EC50 of 28 nM. One uM human CGRP (8-37) shifted the dose-response curve of rat CGRP to the right (EC50 = 257 nM) without depressing the maximal response. Salmon calcitonin (1 nM-1 microM) did not significantly modify total GAG accumulation in FSC cultures. Collagen type I and III production was not significantly affected by either CGRP or calcitonin in FSC cultures. These findings suggest that peripheral sensory neuropeptides may modulate liver fibrogenesis.     

基因表达常规分析方法概述

在基因表达的分析中,RNA转录的稳态水平是检测细胞株和组织的基因表达活性的最方便的参数之一.本概述了用于检测mRNA丰度的常规分析方法,即Northern印迹、RNase保护试验和实时定量PCR技术,特别是为对数量不多的基因进行表达情况的测定时提供了较为实用的方法.