Changes in the infectivity, pyruvate kinase activity, acid phosphatase activity and P-glycoprotein expression in glibenclamide-resistant Leishmania mexicana

We previously demonstrated susceptibility of Leishmania sp. to glibenclamide, a K⁺-ATP transport blocker which interacts with members of the superfamily of adenosine 5′ triphosphate-binding cassette transporters. In order to characterize the molecular differences between a sensitive Leishmania strain, NR(Gs), and an experimentally selected glibenclamide-resistant strain, NR(Gr), specific biochemical and functional parameters have been evaluated both in the wild type and in the resistant strain. Most noteworthy, NR(Gr) exhibit an increased expression of P-glycoprotein and a decreased activity of functional key enzymes such as acid phosphatase, a prominent virulent factor of the parasite, and pyruvate kinase, a key control enzyme for both carbohydrate and protein metabolism. The specific biochemical, metabolic and functional changes observed in the resistant strain correlated with a reduced infectivity of stationary phase NR(Gr) in J774 macrophages and suggested a mechanism to overcome the effect of glibenclamide. Received: 21 January 2000 / Accepted: 1 March 2000     

先天性单侧输精管缺如患者CFTR基因突变研究

目的 探讨囊性纤维跨膜转运调节物（ｃｙｓｔｉｃ ｆｉｂｒｏｓｉｓ ｔｒａｎｓｍｅｍｂｒａｎｅ ｃｏｎｄｕｃｔａｎｃｅ ｒｅｇｕｌａｔｏｒ,ＣＦＴＲ）基因在中国人先天性单侧输精管缺如（ｃｏｎｇｅｎｉｔａｌ ｕｎｉｌａｔｅｒａｌ ａｓｅｎｃｅｏｆ ｔｈｅ ｖａｓ ｄｅｆｅｒｅｎｓ,ＣＵＡＶＤ）患者中的突变频率及热点。方法 应用聚合酶反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）、３银染技术及ＰＣＲ产物直接序     

Antigen presentation in SARS-CoV-2 infection: the role of class I HLA and ERAP polymorphisms

Given the highly polymorphic nature of Human Leukocyte Antigen (HLA) molecules, it is not surprising that they function as key regulators of the host immune response to almost all invading pathogens, including SARS-CoV-2, the etiological agent responsible for the recent COVID-19 pandemic. Several correlations have already been established between the expression of a specific HLA allele/haplotype and susceptibility/progression of SARS-CoV-2 infection and new ones are continuously emerging. Protective and harmful HLA variants have been described in both mild and severe forms of the disease, but considering the huge amount of existing variants, the data gathered in such a brief span of time are to some extent confusing and contradictory. The aim of this mini-review is to provide a snap-shot of the main findings so far collected on the HLA-SARS-CoV-2 interaction, so as to partially untangle this intricate yarn. As key factors in the generation of antigenic peptides to be presented by HLA molecules, ERAP1 and ERAP2 role in SARS-CoV-2 infection will be revised as well.     

丹皮酚对囊性纤维化跨膜电导调节因子氯离子通道的激活作用

目的:研究丹皮酚对依赖于3’,5’-环腺苷酸(cAMP)的囊性纤维化跨膜电导调节因子(CFTR)Cl-通道的激活作用。方法:利用Cl-通道细胞荧光测定模型进行测定丹皮酚对CFTR介导的I-内流速度。结果:丹皮酚对野生型和ΔF508突变型CFTR Cl-通道均具有激活作用,而对G551D突变型CFTR Cl-通道无激活作用。丹皮酚在发挥激活作用时具有快速、可逆的特点并依赖于腺苷环化酶激动剂Forskolin的存在。初步确定了丹皮酚通过直接与CFTR结合发挥作用。结论:研究提示CFTR可能是某些中药降压作用药靶之一。此外,本实验为传统中药的降压作用的分子药理学机制作了初步探讨。     

稳心颗粒对急性心肌缺血的左心室电生理特性的影响

目的研究步长稳心颗粒对家兔缺血心肌左心室内、外膜电生理特性的影响,探讨该药抗心律ar-rhythmia失常的机制。方法在急性缺血条件下,应用浮置玻璃微电极microelectrode记录技术,记录用药前后家兔左室游离壁free wall内、外膜心肌细胞跨膜动作电位transmembrane action potential(TAP),观察动作电位时程ac-tion potential duration(APD)、心室跨壁tanswall复极离散度repolarization dispersion(TDR)的变化。结果①左心室楔形cuneiform组织块停止灌注perfusion后,用药组内、外膜心肌细胞的动作电位时程(APD)缩短,停止灌注时间5 min,10 min,15 min时,内、外膜心肌细胞的动作电位时程逐渐缩短,外膜缩短程度大于内膜(P<0.01)。②急性缺血情况下,用药组的心室跨壁复极离散度(TDR)小于对照组,当停止灌注5 min时,对照组的TDR为(37±12)ms,步长用药组为(30±10)ms(P<0.05)。结论步长稳心颗粒具有抗心肌缺血作用,使缺血情况下左室TDR缩短。     

Silymarin enhanced cytotoxic effect of anti-Fas agonistic antibody CH11 on A375-S2 cells

Silymarin is a polyphenolic flavonoid from milk thistle (Silybum marianum), which has anti-inflammatory, cytoprotective as well as antioxidant effects. Our previous study demonstrated that silymarin has anti-apoptotic effect against UV irradiation. In this study, we assessed the effect of silymarin on anti-Fas agonistic antibody CH11-treated human malignant melanoma, A375-S2 cells. Pretreatment with silymarin (3 × 10^- 4 mol/L) significantly induced cell apoptosis in CH11-treated A375-S2 cells. Mitochondrial transmembrane potential (ΔΨ_m) was also down-regulated by silymarin pretreatment. Caspase-8, -9, -3 and pan-caspase inhibitors partially reversed silymarin-induced apoptosis of CH11-treated cells. The expression of Fas-associated proteins with death domain (FADD), a downstream molecule of the death receptor pathway, was increased by silymarin pretreatment, followed by cleavage of procaspase-8, whose activation induced cell apoptosis. Moreover, cleavage of procaspase-3 and digestion of its substrate, the inhibitor of caspase-activated DNase (ICAD), were also increased by silymarin pretreatment. These results suggested that silymarin could also exaggerate the apoptotic effect of anti-Fas agonistic antibody CH11 on A375-S2 cells.     

Emission ratiometry for simultaneous calcium and action potential measurements with coloaded dyes in rabbit hearts: reduction of motion and drift

INTRODUCTION: Optical measurements of the cardiac calcium transient (Ca) and transmembrane action potential (AP) may be performed simultaneously with emission ratiometry to lessen motion artifacts and photobleaching effects. We examined changes in emission spectrum in perfused rabbit hearts coloaded with Rh237 and a green-emitting Ca dye (Fluo-4 or Oregon Green BAPTA 1) to determine wavelength bands for emission ratiometry and to test whether ratiometry reduces motion artifacts and drift. METHODS AND RESULTS: A 488-nm laser illuminated hearts while a spectrofluorometer collected fluorescence from 489 to 838 nm at 1 kHz. Ratiometry with the Ca- and AP-insensitive emission band 663 to 685 nm (IS) as denominator and the Ca-sensitive band 510 to 532 nm as numerator lessened motion artifacts, which was quantified as a 1.4-fold increase in relative amplitude of the Ca (P < 0.05). Ratiometry with the AP-sensitive band 772 to 794 nm as denominator and the IS as numerator produced a 1.7-fold increase in relative amplitude of the AP (P < 0.05). The ratiometry decreased photobleaching-dependent drift by a factor of 0.6 (P < 0.05) for Ca and 0.45 (P < 0.05) for AP. CONCLUSION: Simultaneous Ca and AP emission ratiometry reduces motion artifacts and drift in hearts with coloaded dyes.     

NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor

The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using ¹²⁵I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.     

Interference with the formation of the epididymal microenvironment-a new strategy for male contraception？

The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Recent work in our laboratory has shown that this protein is also expressed by developing germ cells indicating a role of this protein in spermatogenesis. In view of the fact that the CFTR gene has a far reaching and widespread effect on human reproduction, understanding the role of CFTR in the male reproductive tissues and its intervention by pharmacological agents can open a new avenue of research into the development of novel male contraceptives. ( Asian J Androl 2000 ; 2 : 39 - 45 )     

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Emodin

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time-and dose-dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (Δ Ψ _m), followed by the activation of caspase–9 and caspase–3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide-scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, Δ Ψ _mdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.