Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro.

BACKGROUND: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS: Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS: H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS: The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.     

Thyrotrophin-Releasing Hormone Raises Cytosolic Free Calcium Concentration in Human Adenomatous Somatotrophs and Corticotrophs; Comparison with in vivo Responsiveness to Thyrotrophin-Releasing Hormone in Patients with Acromegaly or Cushing's Disease

The effect of thyrotrophin-releasing hormone (TRH) on intracellular free Ca²⁺ concentration, [Ca²⁺)i, was investigated with the fluorescent dye fura-2 in cell suspensions obtained from 13 human growth hormone-secreting adenomas and 6 adrenocorticotrophin-secreting adenomas. Preoperatively, 9 out of 13 acromegalic patients showed a positive growth hormone response to TRH administration while none of the 6 patients with Cushing's disease had a plasma adrenocorticotrophin increase after TRH injection. In all the growth hormone-secreting adenomas the addition of TRH (100 nM) caused a significant rise in [Ca²⁺]i (from a resting level of 133±40 (±SD) to a value of 284±119 nM at 100 nM TRH, n = 42; P<0.001). The transient induced by TRH was found to have a dual origin, one due to Ca²⁺ mobilization from intracellular stores which was maintained in presence of EGTA (3mM) and verapamil (10 μM) and a plateau phase due to Ca²⁺ influx from the extracellular media. Somatostatin (0.1 μM) lowered both resting [Ca²⁺]i and TRH-induced transients. The effect of gonadotrophin-releasing hormone on [Ca²⁺]i was evaluated on cell suspensions obtained from 6 growth hormone-secreting adenomas. Gonadotrophin-releasing hormone (100 nM) caused a marked rise in [Ca²⁺]i (from 179±25 to 283±15nM) on the cell suspension obtained from the only in vivo responsive adenoma while it was ineffective in the remaining 5. Although TRH was ineffective in modifying plasma adrenocorticotrophin levels in all patients with Cushing's disease, in 5 out of 6 tumors the addition of 100 nM TRH caused a significant rise in [Ca²⁺]i (from 102.5 ± 36 to 163±66 nM, n = 22; P < 0.005). However, the effect of TRH on [Ca²⁺]i was significantly lower than that caused by arginine vasopressin, a physiological stimulator of adrenocorticotrophin release ([Ca²⁺]i values; 145±78 nM at 100 nM TRH versus 300±140 at 10 nM arginine vasopressin, n = 15; P<0.05). Moreover, the effect of arginine vasopressin on [Ca²⁺]i was detectable at concentrations as low as 0.1 nM while TRH was effective at concentrations higher than 1 nM. By contrast, gonadotrophin-releasing hormone was ineffective in increasing [Ca²]i in all the adrenocorticotrophin-secreting adenomas studied. Collectively, these data indicate that sensitivity to TRH is present in almost all the growth hormone- and adrenocorticotrophin-secreting adenomas independently of the responsiveness of the individual patients to the peptide.     

TNF-α和低氧对胰腺癌细胞表达VEGF-A、C的调节

目的:探讨细胞活素肿瘤坏死因子-α(TNF-α)、SNAP(S-Nitroso-Nacetyl-penicillamine)对胰腺癌细胞产生血管内皮生长因子A、C(VEGF-A、C)的调节.方法:用Northern杂交和Western杂交法分析6种人胰腺癌细胞株中VEGF-A、C基因和蛋白的表达;以TNF-α或SNAP刺激其中两个细胞株后用逆转录-聚合酶链式反应技术(RT-PCR)分析其VEGF-A、C基因的表达.结果:Northern杂交法显示这6种胰腺癌细胞株均有4.1kb VEGF-A基因和2.4kb VEGF-C基因的表达;Western杂交法显示它们均有分子量为43kD的VEGF-A蛋白质和分子量为55kD的VEGF-C蛋白质的表达.RT-PCR分析法显示:TNF-α使细胞株COLO-357产生VEGF-A、VEGF-C mRNA分别减少约1～2.5倍、1～2倍,使细胞株CAPAN-1产生VEGF-A、VEGF-C mRNA分别减少约1倍、1.6～2.5倍;而SNAP刺激细胞株COLO-357产生VEGF-A mRNA增加约5倍,刺激细胞株CAPAN-1产生VEGF-A mRNA增加约4倍,但对这两种细胞株产生VEGF-C mRNA均无明显刺激作用.结论:细胞活素TNF-α和低氧通过调节血管内皮生长因子A、C的表达而影响胰腺癌细胞的生物学特性,抑制癌细胞的增殖,促进其凋亡、死亡或进展、恶化.     

Synaptic organization of cortico-cortical connections from the primary visual cortex to the posteromedial lateral suprasylvian visual area in the cat

The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.     

FEEDBACK CONTROL OF A HEAT DIFFUSION SYSTEM WITH TIME-DEPENDENT SPATIAL DOMAIN

The problem of controlling the temperature distribution in a solid cylinder whose length varies with time and with one end in contact with a constant temperature medium is considered. This problem is motivated from that of controlling the temperature and thermal gradient inside a crystal pulled from a melt by the Czochralski method. Boundary feedback controls are derived by considering the time rate of change of a cost functional involving the deviations of both the solid temperature and its gradient from their desired values. The derived feedback controls consist of spatially distributed proportional-plus-rate and lag compensators and a non-linear feedback control involving the temperature gradient at the cylinder surface and the velocity of the spatial domain boundary. The resulting feedback-controlled system has the property that the cost functional along any motion decreases monotonically to zero with time. A numerical scheme for solving the partial differential equation of the feedback-controlled system is proposed. Typical numerical results on the dynamic behaviour of the feedback-controlled system obtained by means of the proposed scheme are presented.     

Raf-1 kinase,epidermal growth factor receptors,and mutant ras proteins in colonic carcinomas

Epidermal growth factor receptors (EGFR) andras mutations are known to play a significant role in controlling cell growth and tumor promotion. Both of them transmit mitogenic signals to the nucleus by activation of Raf-1 kinase. In this study, the expression of EGFR and mutant Ras proteins, and, for the first time, the expression, phosphorylation and kinase activity of Raf-1 kinase have been determined in paired samples of colorectal cancer and mucosa. The tumor and mucosa samples did not differ significantly with regard to Raf-1 kinase content and activity. A major difference between tumors and mucosa was found, however, in the phosphorylation of Raf-1. Most of the mucosa samples (13/20), but only 1/20 of the cancer samples, contained hyperphosphorylated Raf-1. EGFR were significantly (p=0.0025) decreased in the tumors. The decreased phosphorylation of Raf-1 in colonic carcinomas could be the result of activation of Raf-1 phosphatases or inactivation of kinases phosphorylating Raf-1. New forms of treatment based on EGFR overexpression do not seem to be suitable for the majority of colonic cancers.This work was supported by the state of Baden-Württemberg (Verbundforschungsprojekt: Aufklärung von Mechanismen der Tumorentstehung und Tumorabwehr).     

The growth of the second underpotentially deposited silver layer on Pt(100)

The electrodeposition of Ag on Pt(100)-(1 × 1) in perchlorate electrolyte was studied by means of time-resolved in situ scanning tunnelling microscopy (STM) and cyclic voltammetry. One monolayer of Ag is deposited underpotentially (upd) ca. 500 mV positive of the Ag⁺/Ag equilibrium potential. Several millivolts positive of the equilibrium potential, a second well defined upd layer forms. Its growth was observed to proceed via island formation and coalescence. This process occurs in two separate stages that manifest themselves in voltammetric peaks as well as in the STM images.