低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

新生大鼠下丘脑CRF和AVP神经元对急性低氧的应答

目的:观察肾上腺摘除新生大鼠下丘脑促肾上腺皮质激素释放激素(CRF)和精氨酸加压素(AVP)神经元对急性低氧的应答.方法:在低压氧舱中模拟高海拔低氧,用放免法测定AVP和CRP含量.结果:新生大鼠暴露于急性低氧环境下(模拟5 000 m和7 000 m海拔高度,24 h),其下丘脑CRP在3 d和7 d龄大鼠中无明显变化,但14d、21 d和28 d时低于对照;下丘脑AVP在3 d大鼠中亦无变化,但14 d时低于对照,7 d、21 d及28 d时高于对照.两者对低氧的应答模式随日龄而变化.摘除肾上腺后,14 d、21 d及28 d大鼠下丘脑CRF和AVP含量均显著低于同龄完整大鼠,此时暴露于急性低氧环境下,CRF和AVP无进一步的变化.结论:摘除肾上腺抑制下丘脑CRF和AVP的发育,影响它们对低氧应激的正常应答.     

不同环境温度对有机磷杀虫剂毒死蜱引起大鼠体温变化的影响

目的: 观察不同环境温度对有机磷杀虫剂毒死蜱(CHP)引起大鼠体温变化的影响.方法: 用数字体温仪测量大鼠的结肠和尾部皮肤温度,观察25℃、16℃和32℃环境温度对口饲CHP引起结肠温度和尾部皮肤温度变化的影响.结果: 给置于25℃和16℃环境中的大鼠口饲CHP (20 mg·kg-1) 后,均可引起结肠温度明显的降低,尾部皮肤温度明显升高;但32℃环境中大鼠口饲CHP后却引起了明显的升温作用,而尾部皮肤温度无明显影响.结论: CHP对大鼠体温的影响与环境温度有关.     

Regulation of polymorphonuclear leukocyte apoptosis: role of lung endothelium-epithelium bilayer transmigration

Delayed polymorphonuclear leukocyte (PMN) apoptosis exacerbates acute lung injury. To reach the alveolar spaces, PMNs must migrate across both pulmonary endothelial and epithelial cell layers. We hypothesized that transmigration across the endothelium-epithelium bilayer suppresses PMN apoptosis and sought to elucidate the underlying mechanisms. PMNs freshly isolated from normal volunteers were allowed to migrate across polycarbonate membranes alone or membranes coated with a bilayer of human lung endothelial and epithelial cells. After migration toward different chemoattractants (IL-8, formyl-Met-Leu-Phe, or leukotriene B(4)), PMN apoptosis and caspase activities were assessed by annexin V, histology, and enzymatic assays, respectively. Messenger RNA and specific protein expression in three receptor ligand-mediated, apoptosis-inducing pathways (Fas, TNF-alpha, and TNF-related apoptosis-inducing ligand) were further examined by gene array, RT-PCR, flow cytometry, and Western blot analyses. The data demonstrated that transbilayer migration suppressed PMN apoptosis, and this effect was not chemoattractant type specific. Kinetic analyses further showed that the delay of apoptosis was sustained to at least 18 h. Transbilayer migration caused significant decreases in caspase (-3, -8, and -9) activities. The changes in apoptosis-related gene expression support the survival role of transbilayer migration. Furthermore, the reduced apoptosis was correlated with downregulation of Fas ligand and TNF receptor 1 expression. Our data reveal that migration across a lung endothelium-epithelium bilayer suppresses PMN apoptosis. The decreased activity and/or expression of proapoptotic proteins may provide possible targets for the regulation of inappropriate delay in PMN apoptosis during lung inflammation and injury.     

Cellular signals activating muscle proteolysis in chronic kidney disease: a two-stage process

Muscle atrophy is a prominent feature of catabolic conditions and in animal models of these conditions there is accelerated muscle proteolysis that is dependent on the ubiquitin-proteasome system. However, ubiquitin system cannot degrade actomyosin or myofibrils even though it rapidly degrades actin or myosin. We identified caspase-3 as the initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils. In rodent models of catabolic conditions, we find that caspase-3 is activated to cleave muscle proteins and actomyosin to fragments that are rapidly degraded by the ubiquitin system. This initial proteolytic step in muscle can be recognized because it leaves a footprint of a characteristic 14-kDa actin band. Stimulation of caspase-3 activity depends on activation of phosphatidylinositol 3-kinase. When we suppressed this enzyme in muscle cells, protein breakdown increased as did the expression of caspase-3. In addition, there was increased expression of E3-ubiquitin-conjugating enzymes that are involved in muscle proteolysis, atrogin-1/MAFbx and MuRF1. Thus, when phosphatidylinositol 3-kinase activity is low in muscle cells or rat muscle, both caspase-3 and the ubiquitin-proteasome system are stimulated to degrade protein. Additional investigations will be needed to define the cell signaling processes that activate muscle proteolysis in uremia and catabolic conditions.     

Langmuir monolayer approaches to protein recognition through molecular imprinting

Surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM) have been employed to investigate ferritin adsorption to binary surfactant monolayers of cationic dioctadecyldimethylammonium bromide (DOMA) and non-ionic methyl stearate (SME). Surfactant molar ratios, miscibility, and lateral mobility were controlled to define the number, size, and distribution of "binding sites" for ferritin, which under the low ionic strength conditions investigated, adsorbed to the monolayers predominantly through electrostatic interactions. Successive adsorption/desorption cycles revealed that fluid monolayers, capable of laterally restructuring during the initial protein adsorption event, bound up to 60% more ferritin (dependent on SME:DOMA ratios) as compared to monolayers that were immobilized on a hydrophobic support during this first adsorption step. The enhanced binding of ferritin to fluid monolayers was accentuated in films having non-ionic SME as the principal component. These findings support the premise that the surfactants reorganize to form favorable interactions with an adsorbing protein, leading to protein specific charge patterns, or templates, in the films. Template assessment, however, was complicated by the presence of an irreversibly bound protein fraction, which AFM revealed to be locally ordered protein clusters.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

New dual inhibitors of EGFR and HER2 protein tyrosine kinases

A novel series of dual EGFR and HER2 inhibitors based on the pyrrolo[2,1-f][1,2,4]triazine nucleus is described. A general route toward their synthesis, which enables functionalization at multiple sites, has been developed. Biological evaluation in enzymatic and cell-based assays has identified a series of C-6 carbamates with potent biochemical and cellular activities.     

Synthesis and SAR of diazepine and thiazepine TACE and MMP inhibitors

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-alpha release. Oral activity in the mouse LPS model of TNF-alpha release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20.     

The ligand-dependent interaction of mineralocorticoid receptor with coactivator and corepressor peptides suggests multiple activation mechanisms

We investigated the coregulator (coactivator and corepressor) interactions with the mineralocorticoid receptor (MR) that lead to activation and inhibition of the receptor in the presence of agonist and/or antagonist. Our results indicate that MR ligand binding domain (LBD) interacts strongly with only a few specific coactivator peptides in the presence of the agonist aldosterone and that these interactions are blocked by the antagonist eplerenone. We also discovered that cortisol, the preferred physiological ligand for the glucocorticoid receptor in humans, is a partial MR agonist/antagonist, providing a possible molecular explanation of the tissue-selective effects of glucocorticoids on MR. However, when we examined the coactivator and corepressor peptide interactions in the presence of cortisol, we found that MR bound with cortisol or aldosterone interacted with the same set of peptides. Thus, the partial agonism shown by cortisol is unlikely to be the result of differential interaction with known coactivators and corepressors. On the other hand, we have identified coactivator binding groove mutations that are critical for cortisol activation but not for aldosterone activation, suggesting that the two steroids induce different MR LBD conformations. In addition, we also show that cortisol becomes full agonist when S810L mutation is introduced in the LBD of MR. Interestingly, MR antagonists, such as eplerenone and progesterone, become partial agonist/antagonist of S810L but are still able to recruit LXXLL peptides to the mutant receptor. Together, these findings suggest a model to explain the MR activation by various ligands.