Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Single-step nested polymerase chain reaction for detection of different genotypes of hepatitis C virus

Hepatitis C virus (HCV) exhibits considerable sequence variability and circulates in the blood at extremely low levels. Current methods for detecting HCV RNA are based mostly on nested polymerase chain reaction (PCR), in which part of the first amplification product is reamplified in the second tube by an internal primer pair. A novel nested PCR method was developed in which the two successive amplification processes are carried out in the same tube with a single step of physical manipulation. Careful selection of highly conserved sequences of the 5′ noncoding region as primers enabled successful detection of all three major genotypes circulating in France, including the one with variation in this region. Retaining high sensitivity of the conventional nested PCR, the novel method reduced greatly the risk of carry-over contaminations. It was also cost- and time-saving. The one-step nested PCR method is especially suitable for routine diagnosis of HCV infection in clinical laboratories. © 1995 Wiley-Liss, Inc.     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

180例慢性粒细胞白血病骨髓染色体畸变及意义

目的　研究骨髓染色体畸变与慢性粒细胞白血病 (慢粒 )不同病期的相关性及其临床意义。方法　采取新鲜骨髓 ,进行短期培养 (48小时或 12小时预加秋水仙素培养 )制备染色体标本 ,分析 180例慢粒患者慢性期、加速期、急变期骨髓染色体的变异情况。结果　 180例慢粒患者 ,其中 16 4例Ph染色体阳性 (Ph ) ,占 91 1% ;16例Ph染色体阴性(Ph - ) ,占 8 9%。慢性期 133例Ph ,占总数的 73 9% ;加速期 9例Ph ,占总数 5 0 % ;急变期 2 2例Ph ,占总数12 2 %。 16 4例Ph 慢粒患者中 2 5例 (15 2 % )伴有额外染色体异常 ,其中慢性期占 4 2 7%、加速期占 2 4 %、急变期占8 5 3%。结果表明随着病情的进展 ,Ph 阳性率明显增高 ,其它染色体异常率也明显增高。结论　骨髓染色体畸变与慢粒白血病的病程进展、疾病预后有着十分重要的相关性 ,随着病情的演变 ,骨髓染色体畸变趋于复杂化。进行染色体分析对临床诊断及治疗慢粒具有十分重要的指导意义。     

Persistent infection of SARS coronavirus in colonic cells in vitro

Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.     

骨形成蛋白-7对人肾小管上皮细胞株HK-2凋亡的影响

研究不同浓度的骨成形蛋白 7(BMP 7)对人近曲小管上皮细胞株HK 2凋亡的影响并初步探讨其可能的机制。应用Hoechst 332 5 8荧光染色、DNA电泳检测凋亡细胞的形态学和生化改变 ,流式细胞仪定量检测细胞的凋亡率 ,并观察凋亡特异性蛋白酶Caspase 3活性变化的情况。结果显示 ,HK 2细胞加入 1 0ng/mlTGF β处理 2 4h后 ,可明显抑制细胞生长并诱导细胞凋亡 ,呈现明显的凋亡形态学改变 ,胞核或核质内可见浓度致密的颗粒状荧光 ,DNA电泳呈现典型的“梯形条带” ,而预先加入 5 0ng/ml、 1 0 0ng/mlBMP 7共处理 2 4h后 ,可减轻TGF β对细胞的抑制作用 ,凋亡细胞亦减少 ,Caspase 3蛋白酶活性明显下降 ,细胞凋亡率下降。提示BMP 7对人肾小管上皮细胞凋亡的具有抑制作用 ,可能是通过抑制Caspase蛋白酶活性而实现的     

Detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum: comparison with results of other viral markers

A capture enzyme-enhanced chemiluminescence immunoassay (ECLIA) based on three specific monoclonal antibodies to detect the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in the serial serum samples from SARS patients was developed. The anti-SARS-CoV IgG and the viral RNA were also detected in the sera by ELISA and RT-PCR, respectively. During the first 10 days after onset, anti-SARS-CoV IgG, SARS-CoV RNA and the N protein were detected in 21.4, 42.9, and 90% of the patients’ sera, respectively. The detection rate of the N protein during days 11–15 of the disease was still significantly higher than those of anti-SARS-CoV IgG and SARS-CoV RNA. The data demonstrated that detection of the N protein with the capture ECLIA appears to be more useful than detection of other viral makers for rapid diagnosis of SARS in patients.     

散发性帕金森病患者γ-synuclein基因的研究

目的 探讨γ-synuclein基因C243G和A377T多态在散发性帕金森病(Parkinson’s disease，PD)的疾病易感性中的意义。方法 采用聚合酶链反应-限制性片段长度多态法，对上海汉族人中145例PD患者和184名正常人进行γ-synuclein基因C243G和A377T多态分型，并作与PD的遗传关联研究。结果 PD患者与正常对照间γ-synuclein基因C243G和A377T多态分布的差异无显著性，两种多态与PD亦无关联(P值均大于0．05)。结论 上海地区汉族人群中γ-synuclein基因C243G和A377T多态与散发性PD无关。     

Semen parameters and testicular pathology in men with testicular cancer and contralateral carcinoma in situ or bilateral testicular malignancies

We evaluated 14 patients with bilateral testicular tumour, one-sided tumour and contralateral carcinoma in situ (CIS) of the testis or testis tumour in single testis with respect to their fertility. We analysed semen parameters, serum hormones [follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone], testicular sonography, testicular volumes and testicular histology prior to further anti-cancer treatment. Ten out of 14 patients showed normal or reduced sperm concentrations, while 4/14 patients were azoospermic. Serum FSH levels showed a significant negative correlation with sperm concentrations in patients with testicular malignancies (r = -0.64, P = 0.025). Testicular volumes revealed a significant positive correlation with semen parameters in patients with testes that were affected by CIS (r = 0.733, P = 0.038). We conclude that even bilateral testicular cancer and/or CIS do not preclude fertility and, therefore, patients should be offered andrological investigation and therapy, including possibly surveillance strategy or the chance for cryopreservation of the semen prior to further treatment in order to preserve their chances for paternity.