Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

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Endogenous adenosine mediates coronary vasodilation during exercise after K(ATP)+ channel blockade.

The mechanism of coronary vasodilation produced by exercise is not understood completely. Recently, we reported that blockade of vascular smooth muscle K(ATP)+ channels decreased coronary blood flow at rest, but did not attenuate the increments in coronary flow produced by exercise. Adenosine is not mandatory for maintaining basal coronary flow, or the increase in flow produced by exercise during normal arterial inflow, but does contribute to coronary vasodilation in hypoperfused myocardium. Therefore, we investigated whether adenosine opposed the hypoperfusion produced by K(ATP)+ channel blockade, thereby contributing to coronary vasodilation during exercise. 11 dogs were studied at rest and during exercise under control conditions, during intracoronary infusion of the K(ATP)+ channel blocker glibenclamide (50 micrograms/kg per min), and during intracoronary glibenclamide in the presence of adenosine receptor blockade. Glibenclamide decreased resting coronary blood flow from 45 +/- 5 to 35 +/- 4 ml/min (P < 0.05), but did not prevent exercise-induced increases of coronary flow. Glibenclamide caused an increase in myocardial oxygen extraction at the highest level of exercise with a decrease in coronary venous oxygen tension from 15.5 +/- 0.7 to 13.6 +/- 0.8 mmHg (P < 0.05). The addition of the adenosine receptor antagonist 8-phenyltheophylline (5 mg/kg intravenous) to K(ATP)+ channel blockade did not further decrease resting coronary blood flow but did attenuate the increase in coronary flow produced by exercise. This was accompanied by a further decrease of coronary venous oxygen tension to 10.1 +/- 0.7 mmHg (P < 0.05), indicating aggravation of the mismatch between oxygen demand and supply. These findings are compatible with the hypothesis that K+ATP channels modulate coronary vasomotor tone both under resting conditions and during exercise. However, when K(ATP)+ channels are blocked, adenosine released from the hypoperfused myocardium provides an alternate mechanism to mediate coronary vasodilation in response to increases in oxygen demand produced by exercise.     

Infliximab treatment influences the serological expression of matrix metalloproteinase (MMP)-2 and -9 in Crohn's disease

BACKGROUND: Matrix metalloproteinases (MMPs) are actively involved in the pathogenesis of Crohn's disease (CD). We assessed the effect of the anti-tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody infliximab on the in vitro and in vivo expression of MMP-2 and MMP-9 in CD. METHODS: Infliximab-treated fistulizing (n = 10) or active disease (n = 7) CD patients, from an in-house study, and fistulizing CD patients (n = 42) and active CD patients (n = 24) from 2 placebo controlled studies were evaluated for serum MMP levels and clinical response. Biopsies were evaluated immunohistochemically for the MMPs. Whole blood cultures stimulated with lipopolysaccharide (LPS)/infliximab were evaluated for MMP mRNA and protein levels. RESULTS: Serum MMP-2 levels in CD patients increased during follow-up, similarly in responders and nonresponders, by infliximab. Immunohistochemistry showed no clear MMP-2 change in biopsies. Serum MMP-9 levels, however, showed a consistent pattern of decrease in most CD patients, particularly in those responding, and MMP-9-positive polymorphonuclear leukocytes in biopsies also decreased by infliximab. LPS stimulation of whole blood increased the MMP-9 levels in plasma significantly in CD patients and controls, but infliximab had no effect on the secretion. Long-term LPS stimulation raised leukocyte MMP-9 mRNA levels 16-fold and infliximab inhibited this induction by 80%. CONCLUSIONS: Infliximab treatment increases MMP-2 and decreases MMP-9 in serum of patients with CD, the latter also in the intestine, which extends and confirms our previous ex vivo explants observations. However, these changes were not strictly associated with the response to treatment. The enhanced leukocyte MMP-9 expression in CD seems to be regulated by TNF-alpha.     

An allosteric model of circadian KaiC phosphorylation

In a recent series of ground-breaking experiments, Nakajima et al. [Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, Kondo T (2005) Science 308:414-415] showed that the three cyanobacterial clock proteins KaiA, KaiB, and KaiC are sufficient in vitro to generate circadian phosphorylation of KaiC. Here, we present a mathematical model of the Kai system. At its heart is the assumption that KaiC can exist in two conformational states, one favoring phosphorylation and the other dephosphorylation. Each individual KaiC hexamer then has a propensity to be phosphorylated in a cyclic manner. To generate macroscopic oscillations, however, the phosphorylation cycles of the different hexamers must be synchronized. We propose a novel synchronization mechanism based on differential affinity: KaiA stimulates KaiC phosphorylation, but the limited supply of KaiA dimers binds preferentially to those KaiC hexamers that are falling behind in the oscillation. KaiB sequesters KaiA and stabilizes the dephosphorylating KaiC state. We show that our model can reproduce a wide range of published data, including the observed insensitivity of the oscillation period to variations in temperature, and that it makes nontrivial predictions about the effects of varying the concentrations of the Kai proteins.     

PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury

Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage.     

Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish

Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholino-disrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa(121) and VegfAa(165) isoforms, the VegfAb isoforms VegfAb(171) and VegfAb(210) are not normally secreted when expressed in mammalian tissue culture cells. The Kdrb locus encodes a 1361-amino acid transmembrane receptor with strong homology to mammalian KDR. Combined knockdown of both RTKs leads to defects in vascular development, suggesting that they cooperate in mediating the vascular effects of VegfA in zebrafish development. Both VegfAa and VegfAb can individually bind and promote phosphorylation of both Flk1 (Kdra) and Kdrb proteins in vitro. Taken together, our data support a model in the zebrafish, in which duplicated VegfA and multiple type III RTKs mediate vascular development.     

置管灌洗引流治疗脓肿112例体会

本文报告1987年8月到1992年8月,采用置管灌洗引流接负压瓶的方法治疗各种脓肿共112例,其中:94例单个脓肿,18例为多发性脓肿。上肢脓肿24例,躯干脓肿32例,乳房脓肿17例,臀部脓肿21例,肛旁脓肿14例,下肢脓肿16例。脓腔最大为18.0×17.4cm,最个为5.0×4.8cm。平均灌洗引流时间5天,全部治愈。其与手术切开引流相比较,具有:简便、安全、效果确切;且适合于广大基层医院全面推广。本文还具体介绍了置管灌流引流的方法以及术前、术中、术后的临床处理要点;并提出了拨除灌流管的临床指征。     

Gene duplication of zebrafish JAK2 homologs is accompanied by divergent embryonic expression patterns: only jak2a is expressed during erythropoiesis.

Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2 gene may play a role in leukemia. We have isolated and characterized jak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in cloche and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis.