The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na⁺ channel currents. Single Na⁺ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na⁺ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na⁺ channel is directly modulated by phosphorylation process through A-kinase.     

Changes of interleukin expression correlate with Helicobacter pylori infection and lymph node metastases in gastric carcinoma.

Helicobacter pylori (HP) infection induces expression of IL-8 and IL-10 in benign gastric epithelium. This study compared the expression of cytokines in CD4+ and CD8+ lymphocyte subsets of peripheral blood lymphocytes (PBL), benign mucosal lymphocytes (ML), and tumor infiltrative lymphocytes (TIL) as well as in the benign and malignant epithelial cells of the same patient, with respect to the presence of HP infection, lymph node metastases, and tumor histologic type. The mRNA of the cytokines was measured by a semiquantitative RT-PCR method. The levels were ranked and compared using the Wilcoxon sign-ranked test. Compared with CD8+ ML, the CD8+ TIL expresses higher levels of IL-6 and IL-8 but lower level of IL-4 in patients with lymph node metastases. In patients with HP infection, expression of IL-8 and IL-10 was higher in the gastric carcinoma cells than in the benign epithelial cells while expression of IL-6 and IL-8 were higher in CD8+ TIL than CD8+ ML. Overexpression of IL-8 in HP associated gastric carcinomas suggested that they might have arisen from HP-infected epithelial cells.     

Identification and functional analysis of mutations in the hypocretin (orexin) genes of narcoleptic canines

Narcolepsy is a sleep disorder affecting animals and humans. Exon skipping mutations of the Hypocretin/Orexin-receptor-2 (Hcrtr2) gene were identified as the cause of narcolepsy in Dobermans and Labradors. Preprohypocretin (Hcrt) knockout mice have symptoms similar to human and canine narcolepsy. In this study, 11 sporadic cases of canine narcolepsy and two additional multiplex families were investigated for possible Hcrt and Hcrtr2 mutations. Sporadic cases have been shown to have more variable disease onset, increased disease severity, and undetectable Hypocretin-1 levels in cerebrospinal fluid. The canine Hcrt locus was isolated and characterized for this project. Only one novel mutation was identified in these two loci. This alteration results in a single amino acid substitution (E54K) in the N-terminal region of the Hcrtr2 receptor and autosomal recessive transmission in a Dachshund family. Functional analysis of previously-described exon-skipping mutations and of the E54K substitution were also performed using HEK-293 cell lines transfected with wild-type and mutated constructs. Results indicate a truncated Hcrtr2 protein, an absence of proper membrane localization, and undetectable binding and signal transduction for exon-skipping mutated constructs. In contrast, the E54K abnormality was associated with proper membrane localization, loss of ligand binding, and dramatically diminished calcium mobilization on activation of the receptor. These results are consistent with a loss of function for all three mutations. The absence of mutation in sporadic cases also indicates genetic heterogeneity in canine narcolepsy, as reported previously in humans.     

Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome

Clinical studies have provided ample evidence that high (either systemic or local) levels of vascular endothelial growth factor (VEGF) are associated with several pathophysiological disorders, including hemangiomas. To investigate whether elevated VEGF expression could directly affect these disorders, we created a transgenic (Tg) rabbit model with increased hepatic expression of the human VEGF(165) transgene under the control of the human alpha-antitrypsin promoter. Tg rabbits exhibited marked hepatomegaly, with livers 2.5-fold heavier than those of control rabbits. Histological analysis revealed that the livers of Tg rabbits showed prominent dilation of the sinusoids and formed various-sized blood vessel networks, a feature of diffuse hemangiomas. Immunohistochemical staining revealed that the hepatocytes produced VEGF(165), whereas plasma VEGF(165) was not detected. Furthermore, Tg rabbits suffered from hemolytic anemia, thrombocytopenia and splenomegaly, which was associated with marked extramedullary hematopoiesis. The manifestations of Tg rabbits mimic many of the features of hemangiomatous disorders in humans such as the Kasabach-Merritt syndrome, and therefore this model may be potentially useful for the study of the pathogenesis and complications of hemangiomas as well as the investigation of angiogenesis inhibitors.     

Molecular cytogenetic characterization of nasopharyngeal carcinoma cell lines and xenografts by comparative genomic hybridization and spectral karyotyping

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15p14, 7q11.2q21, and 12q22q24.1. Common losses were detected on 3p12p21 (4/6 cases) and 11q14qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1p25, 7p14p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.     

Latent inhibition of conditioned odor potentiation of startle: a developmental analysis

We conducted a two-part study of age and latent inhibition in the rat. In the first part of the study, rats given odor-shock pairings at 23 or 75 days of age exhibited a potentiated startle response in the presence of the odor the following day. This effect did not occur in rats trained at 16 or 20 days of age. Odor pre-exposure on the day prior to conditioning markedly reduced the odor potentiation of startle effect in 23- and 75-day-old rats but had no effect in 16 and 20-day-olds. In the second part of the study, rats were pre-exposed to the odor at 16 or 20 days of age and then conditioned at 23 days of age. When tested the day after conditioning, these pre-exposed rats exhibited a disruption in the odor potentiation of startle effect. We compare our results with other studies of latent inhibition, and with recent studies on whether conditioned responses are appropriate to the animal's age at training or their age at test.     

三氧化二锑诱导急性早幼粒细胞白血病细胞凋亡的研究

目的 研究锑剂三氧化二锑（Sb2O3)对早幼粒细胞白血病细胞株NB4凋亡的诱导作用，以寻求早幼粒细胞白血病治疗的新方法。方法 采用细胞生长曲线，形态学及硝基四氮唑蓝（NBT）还原试验，判定NB4细胞的生长，分化及功能。采用细胞周期分析和DNA电泳研究细胞凋亡。结果 Sb2O3能诱导早幼粒白血病细胞凋亡，且具有时间，剂量依赖性。结论 Sb2O3能有效地诱导早幼粒白血病细胞凋亡，提示锑剂诱导细胞半亡的疗法，有望成为临床治疗早幼粒细胞白血病的新方法。     

Sodium nitroprusside,a NO donor,modifies Ca2+ transport and mechanical properties in frog skeletal muscle

Recently it has been hypothesized that, in skeletal muscle, NO produced directly by high-frequency stimulation could produce contraction through reactions with thiol groups on the sarcoplasmic reticulum (SR). However, a possible cGMP-mediated relaxing effect, similar to that seen in smooth muscle, has also been demonstrated. We used purified SR preparations and single fibres from frog fast muscles incubated with different concentrations of sodium nitroprusside (SNP) in this study. The results obtained from a long low-frequency stimulation, together with those from a study on Ca2+ transport regulation, showed that the presence of NO precursor induced: an acceleration of the onset of fatigue in single fibres; a decreased vesicular Ca2+ content due to increased Ca2+ release; a shift to open status in SR Ca2+ channels; an increase in SR Ca2+ pump activity. The data presented in this paper seem to indicate that the increased NO in the muscle fibres can influence muscle activity in different ways, perhaps depending on the metabolic status of the muscle and target (filaments, sarcolemma, SR) with which the NO (or its derivatives) acts.     

人肿瘤细胞与正常细胞内微管的PAP免疫酶细胞化学观察

我们利用兔抗微管蛋白抗体和兔抗辣根过氧化物酶(HRP)抗血清制备的HRP—抗HRP(PAP)复合物,建立了微管的PAP免疫酶细胞化学方法。应用此法观察到人食管癌ECa 109、胃癌SGC 7901,乳腺癌MCF 7和成骨肉瘤OS 732细胞间期胞质微管减少或消失,只有大量弥散分布的微管蛋白棕色反应产物,在微管组织中心(MTOC)附近十分密集,而正常成纤维细胞和胎儿胃粘膜上皮细胞间期,都有发达的胞质微管结构(CMTC)。在分裂期,这些肿瘤细胞都显示纺锤体微管,与正常细胞比较无明显差异。本研究应用PAP方法进一步证明,以前用免疫荧光细胞化学方法观察到的人肿瘤细胞间期胞质微管缺陷的特征。除去低温(4℃)或秋水仙酰胺处理后,解聚的CMTC又可恢复,表明本方法与免疫荧光染色法,同样具有很高的特异性。本工作在细胞固定及免疫反应的某些步骤上有所改进。