Further analysis of ATP-mediated activation of K+ channels in renal epitheloid Madin Darby canine kidney (MDCK) cells

ATP activates K⁺ channels by increasing intracellular calcium activity in Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for the involvement of G-proteins and of protein kinase C in the intracellular transmission of these effects. To this end, the effect of ATP on intracellular calcium and K⁺ channel activity has been studied in cells pretreated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or pertussis toxin. The ATP-induced increase of intracellular calcium is not significantly affected by pretreatment with pertussis toxin, is significantly blunted by pretreatment with TPA and is abolished by pretreatment with both pertussis toxin and the phorbol ester. The ATP activation of K⁺ channels is similarly blunted by pretreatment with TPA, but is not abolished by pretreatment with both the phorbol ester and pertussis toxin. Furthermore, the ATP-induced hyperpolarization is not abolished in cells pretreated with both pertussis toxin and TPA. In those cells, ATP may activate K⁺ channels by calcium-independent mechanisms or lead to localized increases of intracellular calcium sufficient to activate the K⁺ channels but escaping detection with fura-2 fluorescence.     

蛙类肠系膜内微小动脉的压力检测

根据Wiederhielm的阻抗平衡压力检测原理,设计了一个改进的伺服零微血管测压系统。利用该系统检测了蟾蜍及蛙肠系膜内微小动脉的压力、获得了各管径级的压力和脉压的参数,并观察了药物的作用,探讨了微动脉内压力的波动特性以及应激情况下压力的骤变式与脉动式的交替,为微循环研究提供一些有价值的现象。     

结核分枝杆菌联合DNA疫苗初免-BCG加强的免疫效果观察

目的:研究并比较结核分枝杆菌免疫保护性抗原DNA(Ag85A和ESAT-6)疫苗联合免疫,BCG免疫以及联合DNA疫苗初免-BCG加强免疫等不同的免疫策略,诱导免疫应答效果观察.方法:健康雌性BALB/c小鼠24只,随机分成PBS 阴性对照组,DNA初免-BCG异源加强组,DNA(Ag85A和ESAT-6)初免DNA同源加强组和BCG阳性对照组,共进行3次免疫,初免2次,最后1次加强,间隔2周1次.PBS组3次均注射PBS 溶液;DNA/BCG组以质粒DNA免疫2次,最后1次以BCG加强免疫;DNA/DNA组3次均以质粒DNA进行免疫;BCG组则注射PBS溶液2次后以BCG免疫.末次免疫后4、6、8周分别分离血清测定总IgG水平,同时分离小鼠脾细胞,体外经TB-PPD刺激后进行淋巴细胞增殖实验(XTT法)并测定脾细胞培养上清中IFN-γ和IL-4水平.结果:DNA/BCG、DNA/DNA、BCG组体外经TB-PPD刺激后均检测到特异性IgG抗体产生,3组平均效价为1:120、1:160、1:80,DNA/DNA组的抗体效价高于另外2组;小鼠脾细胞体外经TB-PPD刺激后,均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,其中DNA/BCG组IFN-γ的分泌水平高于DNA/DNA组和BCG组(P<0.05).结论:联合DNA疫苗初免-BCG加强的免疫策略能在小鼠体内诱导较强的特异性细胞免疫反应,产生高水平的IFN-γ.     

NGF在成年猴脑的分布

为了解NGF在成年猴脑的分布,采用免疫组化SP法对成年猴脑多个冠状位切片进行免疫组化反应。结果证明,NGF阳性反应神经元主要分布于大脑皮质Ⅲ、V层,小脑Purkinje细胞,海马,齿状回,纹状体,脑干网状结构等处。此外,在黑质、舌下神经核、迷走神经背核、前庭神经核、三叉神经核、疑核、下橄榄核也出现NGF阳性反应。在大脑和脑干还观察到NGF阳性胶质细胞。本实验结果表明,在成年猴脑的多个脑区有NGF表达,提示NGF可能涉及猴脑某些神经元及胶质细胞的生理过程。     

Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-γ) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-γ-induced surface expression of class II antigens as well as the IFN-γ-induced up-regulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-γ for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started ≈ 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E₂, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.     

Identification and characterization of neurons with tremor-frequency activity in human globus pallidus

 Many previous studies have demonstrated the existence of neurons with tremor-frequency activity (”tremor cells”) in the thalamus of Parkinson’s disease (PD) patients and these neurons are presumed to play a role in the pathogenesis of tremor. Since a major input to motor thalamus (Voa and Vop) is from the internal segment of the globus pallidus (GPi), neurons with tremor-frequency activity in motor thalamus may receive input from neurons in GPi. The aim of this study was to quantify the characteristics of tremor cells in human globus pallidus. In three PD patients with tremor undergoing microelectrode exploration of the globus pallidus prior to pallidotomy, 228 neurons were sampled, and 28 (12.3%) were identified to fire at the same frequency as the tremor. These ”tremor cells” were located in the ventral portion of GPi. Autocorrelogram analysis of the sampled spike trains of these 28 tremor cells was carried out over sequential 10-s time segments, and autocorrelograms showing maximal oscillatory activity were graded from 0 to 10. Average tremor cell oscillation grades ranged from 6.8 to 7.8, similar to those reported in the MPTP-induced primate model of parkinsonism. The average tremor cell oscillation grade varied between patients, as did the clinical measures of tremor severity. Tremor cells had oscillations in spike discharges at the same average frequency (4.2–5.2 Hz) as the patient’s tremor determined from the electromyogram and accelerometry records of one or more limbs (4.0–5.4 Hz), and the individual values were correlated (r ²=0.73) over the total range (3.7–5.6 Hz). The results of this study demonstrate the presence of neurons with 4–6 Hz tremor-frequency activity in GPi, supporting a role of the globus pallidus in the production of rest tremor in PD patients. Received: 27 February 1996 / Accepted: 18 October 1996     

Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD⁺-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA. Received: 19 January 2000 / 24 March 2000     

In Situ Detection of Apoptosis at Sites of Chronic Bacterially Induced Inflammation in Human Gingiva

Apoptosis is a key phenomenon in the regulation of the life span of terminally differentiated leukocytes. Human gingiva represents an established model to study immune responses to bacterial infection. In this investigation, we used the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) technique to evaluate presence and topographic location of apoptosis-associated DNA damage in human gingival biopsies along with the expression of the p53 and Bcl-2 apoptosis-regulating proteins. Qualitative data analysis showed high densities of cells expressing DNA damage and p53 both within the epithelial attachment to the tooth and in the perivascular infiltrate (infiltrated connective tissue [ICT]) immediately underlying the site of chronic bacterial aggression. Topographic consistency between DNA damage- and p53-positive cells was consistently observed. Quantitative analysis of the ICT showed mean densities of DNA damage- and p53-positive cells of 345 ± 278 and 403 ± 182 cells/mm², respectively. Numerical consistency was confirmed by multivariate regression analysis: densities of DNA damage-positive cells were significantly predicted by densities of p53-positive cells (P = 0.001, r² = 0.84). In the ICT, cells displaying biotinylated DNA nicks were 3.8% ± 2.7% of total cellularity, while p53- and Bcl-2-positive cells represented 4.4% ± 1.7% and 15.4% ± 6.7% of total cells, respectively. It is suggested that p53 expression associated with DNA damage is a prevalent phenomenon in chronically inflamed human gingiva, and that apoptosis may be a relevant process for the maintenance of local immune homeostasis at sites of chronic bacterial challenge in vivo.     

Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinomas (HNSCC) often metastasise to the cervical lymph nodes. It is known for HNSCC as well as other cancers that progression from normal tissue to primary tumour and finally to metastatic tumour is characterised by an accumulation of genetic mutations. DNA methylation, an epigenetic modification, can result in loss of gene function in cancer, similar to genetic mutations such as deletions and point mutations. We have investigated the DNA methylation phenotypes of both primary HNSCC and metastatic tumours from 13 patients using restriction landmark genomic scanning (RLGS). With this technique, we were able to assess the methylation status of an average of nearly 1300 CpG islands for each tumour. We observed that the number of CpG islands hypermethylated in metastatic tumours is significantly greater than what is found in the primary tumours overall, but not in every patient. Interestingly, the data also clearly show that many loci methylated in a patient's primary tumour are no longer methylated in the metastatic tumour of the same patient. Thus, even though metastatic HNSCC methylate a greater proportion of CpG islands than do the primary tumours, they do so at different subsets of loci. These data show an unanticipated variability in the methylation state of loci in primary and metastatic HNSCCs within the same patient. We discuss two possible explanations for how different epigenetic events might arise between the primary tumour and the metastatic tumour of a person.