Localization of tissue plasminogen activator mRNA in adult rat brain

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with ³⁵S UTP-labeled RNA probes derived from a fulllength tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 8% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3–11 % of cells in individual brain areas.     

Decreased antibody-dependent cellular cytotoxicity in minimal change nephrotic syndrome

Secondary IgG response to a tetanus toxoid booster and in vitro measurement of immunoglobulin synthesis, antibody-dependent cellular cytotoxicity (ADCC) and -interferon (IFN-) production were evaluated in 20 healthy controls and in 17 children with minimal change nephrotic syndrome (MCNS), during the acute nephrotic phase and 6 months after remission. Defective responses were observed in all but IFN- production during the acute nephrotic phase; these improved with disease remission. There was a significant correlation between decreases in vitro IgG production and ADCC reaction. These data indicate that defective antibody production is associated with decreased ADCC during the acute nephrotic phase of MCNS.     

Chronic ethanol exposure differentially regulates NOS1 mRNA levels depending on rat brain area

In vitro experiments were performed on brainstem – spinal cord preparations from mouse neonates to compare the noradrenergic regulations of the respiratory network in the control C3H/HeJ strain and the transgenic Tg8 strain which has been created from the C3H/HeJ strain by deletion of the gene encoding monoamine oxidase A (MAOA), the main enzyme for serotonin degradation. In both control and MAOA-deficient strains, we show: (i) that the pontine A5 area exerts a potent inhibitory modulation on the respiratory rhythm generator; (ii) that noradrenaline application induces a tonic phrenic activity; and (iii) that noradrenaline increases the respiratory rhythm. The latter effect is however delayed and weak in the Tg8 strain. Therefore, MAOA-deficiency has only slightly altered the noradrenergic regulations of the respiratory network.     

Tumor genes and their proteins in cytologic and surgical specimens: Relevance and detection systems

Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases. Tumor-related genes can be classified into functional categories. Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation. More recently, three other groups of tumor-related genes have been recognized. They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes. Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response. The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques. These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist. © 1995 Wiley-Liss, Inc.     

Simultaneous detection of fluorescent in situ hybridization and in vivo incorporated BrdU in a human bladder tumour

We have used fluorescent in situ hybridization and simultaneous in vivo bromodeoxyuridine labelling of a solid bladder cancer to examine tumour cell subsets for possible proliferative growth differences. In this dual-labelled preparation, most tumour cell nuclei exhibited monosomy 9, consistent with reported karyotypes of bladder cancer. Incorporated bromodeoxyuridine was visualized with a fluoresceinated antibody in 5-6 per cent of the tumour cells, concordant with S-phase estimates by cell cycle analysis of the flow cytometric DNA histogram. A majority of the bromodeoxyuridine-positive cells also carried the monosomy 9 chromosome abnormality. This is the first report to demonstrate the feasibility of combined in situ hybridization and detection of bromodeoxyuridine incorporated in vivo in human tumour cells in order to provide information on the growth rate of specific subsets of tumour cells identified by chromosomal constitution.     

眼附属器淋巴组织增生性病变的临床病理和分子遗传学特征及其意义

目的 分析眼附属器淋巴组织增生性病变的临床病理特点,探讨其分子遗传学特征及其意义.方法 收集1995-2007年37例眼附属器淋巴组织增生性病变石蜡组织标本(其中5例为反应性增生性病变,32例为淋巴瘤),依据2001年WHO肿瘤分类标准对32例淋巴瘤标本重新诊断分类.采用IgH、MALT1、bcl-6、c-Mye、bcl-2、CCND1、bcl-10、FOXP1双色分离重排探针、IgH/bcl-2双色融合易位探针和18号染色体着丝粒探针,利用间期荧光原位杂交(FISH)的方法 检测眼附属器淋巴组织增生性病变的分子遗传学特点.结果 32例淋巴瘤均为非霍奇金B细胞淋巴瘤.其中,黏膜相关淋巴组织结外边缘区B细胞淋巴瘤(MALT)淋巴瘤28例(87.5%),滤泡性淋巴瘤2例,弥漫性大B细胞淋巴瘤2例.60.7%(17/28)的眼附属器MALT淋巴瘤携带分子遗传学异常.其中,IgH基因断裂1例,但未找到与其发生相互易位的伙伴基因;基因3拷贝者16例,其中MALT1基因、bcl-6基因和c-Myc基因3拷贝的发生率分别为25%(7/28)、43%(12/28)和7%(2/28).16例基因3拷贝病例中,两种基因3拷贝合并存在者5例,其中bcl-6基因合并MALT1基因3拷贝者4例,bcl-6基因合并c-Myc基因3拷贝者1例.进一步研究显示,MALT1基因3拷贝者均存在18号染色体三体.2例滤泡性淋巴瘤都携带t(14;18)(q32;q21)/IgH-bcl-2.2例弥漫性大B细胞淋巴瘤均存在遗传学异常,1例表现为bcl-6基因3拷贝合并18号染色体三体,另1例表现为bcl-6基因3拷贝合并IgH和c-Myc基因双断裂.5例反应性淋巴组织增牛性标本均未见分子遗传学异常.结论 MALT淋巴瘤是眼附属器最常见的淋巴瘤类型;间期FISH有助于淋巴组织增生性病变的良恶性鉴别及淋巴瘤的分类;MALTI基因3拷贝者由18号染色体三体所致;18号染色体三体和bcl-6基因3拷贝(可能为3号染色体三体所致)是眼附属器MALT淋巴瘤常见的分子遗传学异常.     

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.     

Simultaneous electrical and mechanical recording in small isolated perfused arteries

A new method is described for simultaneous measurements of electrical and mechanical activity in an intact segment of a small artery (outside diameter 0·3–0·5 mm). A 7–9 mm segment of rabbit middle-cerebral artery was ligated on a Teflon tube connected to a perfusion circuit. The presence of a plug in the tube, flanked by two orifices, forced the physiological solution to flow in the annular space between the tube and the artery wall. Physiological pressures could thus be attained at low rates of flow, and the pressure, measured upstream, was significantly modifed by the slightest constriction or dilatation of the vascular segment. Electrical recording with glass microelectrodes was performed on a short immobilised portion of the artery. The artery and tube were bathed in a physiological solution at 38°C, but perfusing and incubating solutions did not mix. Spontaneous electrical and mechanical activity of middle cerebral arteries is described, together with modifications induced by vasoactive agents.     

Localization of two cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter in the central nervous system of the rat: in situ hybridization histochemistry and immunohistochemistry

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are proteins that are required for cholinergic neurotransmission. Present knowledge concerning the organization of cholinergic structures has been derived primarily from immunohistochemistry for ChAT. In the present study, we investigated the distribution of mRNAs and the corresponding proteins for ChAT and VAChT by in situ hybridization histochemistry and immunohistochemistry. The patterns of distribution of perikarya containing ChAT mRNA, ChAT protein, VAChT mRNA and VAChT protein were similar in most regions, and co-localization in the same neuron of mRNAs for ChAT and VAChT, that of ChAT mRNA and ChAT protein, and that of VAChT mRNA and VAChT protein were demonstrated. However, in the cerebral cortex and hypothalamus, ChAT-immunoreactive perikarya were present, but they did not contain mRNAs for ChAT and VAChT, and VAChT protein. On the other hand, in the cerebellum, Purkinje cell bodies contained VAChT mRNA and VAChT protein, but they did not contain either ChAT mRNA or ChAT protein. Axon bundles were clearly revealed by immunohistochemistry for ChAT, but they were not detected by that for VAChT. Both ChAT and VAChT antibodies revealed preterminal axons and terminal-like structures. In the forebrain, they were present in the olfactory bulb, nucleus of the lateral olfactory tract, olfactory tubercle, lateral septal nucleus, amygdala, hippocampus, neocortex, caudate-putamen, thalamus and median eminence of the hypothalamus. In the brainstem, they were localized in the superior colliculus, interpeduncular nucleus and some cranial nerve motor nuclei, and further in the ventral horn of the spinal cord. These results indicate strongly that ChAT and VAChT are expressed in most of the cholinergic neurons, and that immunohistochemistry for VAChT is as useful to detect cholinergic terminal fields as that for ChAT.     

Microdialysis: a way to study in vivo release of neurotrophic bioactivity: a critical summary

Microdialysis has been proven to be a valuable tool to study in vivo release of various neurotransmitters in the rat brain. Recently we demonstrated for the first time the release of neurotrophic bioactivity in the brains of awake rats. Neurotrophic factors, however, exist in extremely low concentrations in the brain compared to neurotransmitters, rendering their detection particularly difficult. This review summarizes knowledge about the use of microdialysis for the detection of neurotrophic bioactivity, its limits, and its problems.Abbreviations BDNF Brain-derived neurotrophic factor - EIA Enzyme immunoassay - NGF Nerve growth factor - NT-3 Neurotrophin-3