Evidence for cAMP-independent bTREK-1 inhibition by ACTH and NPS-ACTH in adrenocortical cells

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K⁺ channels that are inhibited by ACTH through cAMP-dependent pathways. In whole cell patch clamp recordings from AZF cells, we found that ACTH may also inhibit bTREK-1 by a cAMP-independent mechanism. When the potent adenylyl cyclase (AC) antagonist 2,5-dideoxyadenosine-3′-triphosphate (2,5-dd-3′-ATP) was applied intracellularly through the patch pipette, bTREK-1 inhibition by the AC activator forskolin was blocked. In contrast, bTREK-1 inhibition by ACTH was unaltered. The selective G_Sα antagonist NF449 also failed to blunt bTREK-1 inhibition by ACTH. At concentrations that produce little measurable increase in cAMP in bovine AZF cells, the O-nitrophenyl, sulfenyl-derivative of ACTH (NPS-ACTH) also inhibited bTREK-1 almost completely. Accordingly, 2,5-dd-3′-ATP at concentrations more than 1000× its reported IC₅₀ did not block bTREK-1 inhibition by NPS-ACTH. These results indicate that ACTH and NPS-ACTH can inhibit native bTREK-1 K⁺ channels in AZF cells by a mechanism that does not involve activation of AC.     

Age-related loss of cardiac preconditioning: impact of protein kinase A

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK_Ca) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK_Ca channels is protein kinase A (PKA).We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent.Young (2-3 months) and aged (22-24 months) Wistar rats were randomised to eight groups (each n = 8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3 × 5min (He-PC). The PKA-blocker H-89 (10 μg/kg) was administered with and without helium (He-PC + H-89, H-89). Furthermore, we tested the effect of direct activation of mK_Ca channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 μg/kg) and aged animals (300 and 1000 μg/kg).He-PC reduced infarct size from 60 ± 4% (Con) to 37 ± 10% (p < 0.05). Infarct size reduction was completely abolished by H-89 (58 ± 5%; p < 0.05), but H-89 alone had no effect (57 ± 2%). NS1619 reduced infarct size in the same concentration in both, young and aged rats (35 ± 6%; p < 0.05 vs. Con and 34 ± 8%; p < 0.05 vs. Age Con). Forskolin in a concentration of 300 μg/kg reduced infarct size in young (37 ± 6%; p < 0.05) but not in aged rats (48 ± 13%; n.s.). In contrast, 1000 μg/kg Forskolin reduced infarct size also in aged rats (28 ± 3%; p < 0.05).He-PC is mediated by activation of PKA. Alterations in PKA regulation might be an underlying mechanism for the age-dependent loss of preconditioning.     

Regulator of G-protein signaling 2 inhibits acid-induced mucin5AC hypersecretion in human airway epithelial cells

Mucus hypersecretion is a common pathological feature of inflammatory airway diseases. Previous studies have shown that acidic microenvironment of inflamed airways may provoke the pathophysiology of inflammatory airway diseases. However, the acidic-sensing and negative regulatory mechanisms that mediate mucus hypersecretion in inflamed airway remain elusive. Thus, we sought to explore the role of ovarian cancer G-protein-coupled receptor 1 (OGR1) in acid-induced mucin5AC (MUC5AC) hypersecretion in human airway epithelium and the inhibitory effect of regulator of G-protein signaling 2 (RGS2) in this process. We found that airway acidification increased [Ca²⁺]_i, which was required for MUC5AC secretion. Knocking-down OGR1 and G_q with siRNAs and pretreating cells with phospholipase C inhibitor effectively attenuated acid-induced cellular responses. Moreover, the overexpression of wild-type RGS2 attenuated acid-induced cellular responses. In contrast, reducing RGS2 with siRNA enhanced the increases in acid-induced cellular responses. These data suggest that airway acidification can induce MUC5AC hypersecretion through an OGR1-mediated mechanism and RGS2 can inhibit acid-induced MUC5AC hypersecretion at OGR1 receptor level.     

Prognostic value of the stem cell markers CD133 and ABCG2 expression in esophageal squamous cell carcinoma

In the light of increasing evidence supporting cancer stem cells (CSCs) theory, the expression of two stem cell markers, CD133 and adenosine triphosphate-binding cassette superfamily G member 2 (ABCG2), in esophageal squamous cell carcinoma (ESCC) was investigated, and their prognostic values were evaluated. Paraffin-embedded tissue sections of 110 ESCC patients were investigated using Immunohistochemistry. The association of CD133 and ABCG2 expression with clinicopathologic characteristics was analyzed by χ(2) test. Survival analysis was carried out using Kaplan-Meier method and Cox proportional hazards model. CD133 and ABCG2 expression were detected in 27.3% and 15.5% of ESCC patients, respectively. The presence of CD133-positive cancer cells was associated with tumor cell differentiation (P= 0.008) but not significantly related to the survival of ESCC patients (P= 0.085). ABCG2 expression was not associated with clinicopathologic characteristics but was a significant prognostic factor for adverse overall survival of ESCC patients (P= 0.005). The median overall survival time for ESCC patients with and without ABCG2 expression were 21.8 and >49.3 months, respectively. A combined analysis of CD133 and ABCG2 expression did not show that ESCC patients with coexpression of these two markers had a worse prognosis than those with only ABCG2 expression (P= 0.934). Moreover, ABCG2 expression was revealed to be an independent prognostic factor along with tumor node metastasis stage in multivariate analysis (hazard ratio of ABCG2, 3.38; 95% confidence interval, 1.61～7.09; P= 0.001). By survival analysis based on tumor node metastasis stage of ESCC, the association between ABCG2 expression and the patients' prognosis was found significant in the group of relatively early stage (P= 0.005) and marginally significant in the group of relatively late stage (P= 0.058). This is the first time to report the presence of CD133-positive cancer cells in ESCC but not supporting its prognostic value and validity as a CSC marker for ESCC. ABCG2 expression was found to correlate with the survival of ESCC patients, especially those at relatively early stage, suggesting that ABCG2-positive cancer cells may represent a pool of CSCs in ESCC, and relatively early-stage patients with ABCG2 expression may deserve more intensive or targeted therapy.     

RETRACTED: A new test for acromio-clavicolar pathology

Background

A prospective study was established to assess the sensitivity and specificity of the new Saccomanni (SAC) test for isolated AC pathology, and compare with 4 commonly used clinical tests.

Materials and methods

The Saccomanni (Sac) test is essentially the cross-adduction test, with the addition of attempted elevation against resistance. In a positive test, this results in some pain and the inability of the patient to maintain the arm in the adducted and elevated position against resistance. Fifty-eight patients with isolated AC joint symptoms were assessed in random order with the Saccomanni test and 4 other tests. A corticosteroid and local anaesthetic injection was administered into the AC joint space. The Saccomanni test and 4 other tests were then repeated following the injection. After the injection, a symptom free clinical examination was used as a measure of true positive tests.

Study design

Case series.

Results

The SAC test showed a sensitivity of 98% and specificity is 91.7%. All 4 other tests were less sensitive.

Conclusion

The SAC test is a highly sensitive test in patients presenting with isolated AC related symptoms.This study is an innovation for clinical tests in the world. The primary aim of this study was to assess the diagnostic sensitivity of my newly described SAC test. From the present study, it can be concluded that the easy-to use SAC is a highly sensitive test to evaluate AC joint pathology, when compared to other standard tests.

Clinical relevance

Level III, Diagnostic Study of Nonconsecutive Patients.     

胃癌原发灶中上皮间质转化相关因子和CD133的表达及其与临床病理特征和预后的关系

目的研究上皮间质转化（EMT）相关因子Snail、E-cadherin、N-cadherin与胃癌患者临床病理特征、预后及胃癌肿瘤起始细胞表面标志物CD133表达的关系。方法利用Western blot方法检测50例胃癌及癌旁正常胃黏膜组织中EMT相关因子及CD133蛋白的定位及定量表达,分析EMT相关因子及CD133蛋白表达与胃癌患者的临床病理学指标的关系,Spearman等级相关分析EMT相关因子和CD133表达的关系,Kaplan-Meier方法分析EMT相关因子及CD133表达与胃癌患者生存的关系。结果①胃癌组织中Snail、N-cadherin及CD133蛋白表达相对灰度值明显高于其在癌旁正常胃黏膜组织中的表达（Snail：0.599±0.114比0.259±0.108,P=0.020;N-cadherin：0.754±0.154比0.329±0.134,P=0.001;CD133：0.635±0.119比0.485±0.116,P=0.029）,E-cadherin蛋白表达相对灰度值明显低于其在癌旁正常胃黏膜组织中的表达（0.378±0.123比0.752±0.156,P=0.003）。②Snail蛋白、N-cadherin蛋白表达平均相对灰度值在有血管浸润、淋巴管浸润、N3淋巴结转移及肿瘤直径≥5 cm和Ⅲ＋Ⅳ期胃癌患者中的表达明显高于无血管浸润、淋巴管浸润、N0～N2淋巴结转移及肿瘤直径〈5 cm和Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）,而E-cadherin蛋白表达平均相对灰度值在有血管浸润、淋巴管浸润、N3淋巴结转移及Ⅲ＋Ⅳ期胃癌患者中的表达显著低于无血管浸润、淋巴管浸润、N0～N2淋巴结转移及Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）,CD133蛋白表达平均相对灰度值在有淋巴管浸润、N3淋巴结转移、肿瘤直径≥5 cm和Ⅲ＋Ⅳ期胃癌患者中的表达显著高于无淋巴管浸润、N0～N2淋巴结转移、肿瘤直径〈5 cm和Ⅰ＋Ⅱ期的胃癌患者（P〈0.05）。③Snail、N-cadherin蛋白表达与CD133蛋白表达分别均呈正相关（rs=0.278,P=0.048;rs=0.406,P=0.003）,而E-cadherin蛋白表达与CD133蛋白表达呈负相关（rs=-0.504,P=0.000）。④Snail、N-cadherin及CD133蛋白低表达组的生存时间明显长于其高表达者（P〈0.05）,联合EMT相关因子和CD133蛋白表达能够最有效预测患者生存。结论EMT与胃癌肿瘤起始细胞特性之间存在明显相关,并且两者与胃癌的高侵袭的临床病理特征相关,联合EMT相关因子Snail、E-cadherin、N-cadherin与CD133能够最有效预测胃癌患者的预后。     

Assessment of cerebral blood flow and metabolism in vertebrobasilar insufficiency with 133Xenon and 15O inhalation methods

Abstract

Assessment of hemodynamic parameters in patients who had transient basilar symptoms suggesting vertebrobasilar insufficiency requires a systematic and .accurate detection of brainstem or cerebellar infarcts. Our aim was to detect with 133Xenon and ¹⁵0₂ inhalation methods, a low flow state underlying vertebrobasilar insufficiency in a patient who had no impairment of cerebrovascular control related to infarction of brainstem or cerebellum. A woman with intermittent vertebrobasilar symptoms had an angiogram, magnetic resonance imaging and evaluation of hemodynamic parameters with vertebrobasilar circulation with 133Xenon inhalation and ¹⁵0 inhalation methods MRI failed to show any border-zone or territorial infarcts or degenerative disease..Angiographic study showed significant arterial lesion involving vertebrobasilar vessels. A decrease ofblood flow within vertebrobasilar circulation was observed according to 133Xen"on and ¹⁵0 inhalation methods. These preliminary results support the view that significant arterial changes within the vertebrobasilar system might account for a low flow state. ¹⁵ 0 ₂ inhalation method might be in agreement with a previous study performed in vertebrobasilar insufficiency with 133Xenon inhalation method. [Neural Res 1997; 19: 165–168]