Effects of selective NMDA and non-NMDA blockade in the nucleus accumbens on the plus-maze test

Effect of blocking N-methyl-D-aspartic acid (NMDA) and non-NMDA-glutamatergic receptors on performance in the plus-maze was studied in male rats bilaterally cannulated into the nucleus accumbens (Acc). Rats were divided into seven groups that received either 1 microl injections of saline, (+/-)2-amino-7-phosphonoheptanoic acid (AP-7, 0.2, 0.5, or 1 microg) or 2,3 dioxo-6-nitro-1,2,3,4,tetrahydrobenzo-(f)quinoxaline-7-sulphonamide disodium (NBQX, 0.2, 0.5, or 1 microg) 15 min before testing. Time spent in open arm, time per entry, end arrivals, open, closed, and total arm entries, relationship between open-, closed-, and total arm entries, rearing, face-, head-, and body grooming, and number of fecal boli were recorded. Time spent in the open arm increased under AP-7 (0.5 and 1 microg; P<.01) and NBQX (1 microg; P<.05) treatment, whereas time per entry was increased only with AP-7 (1 microg; P<.05). Open arm entries were increased by the intermediate doses of AP-7 (0.5 microg; P<.01) and NBQX (0.5 microg; P<.05); end arrivals were increased by the intermediate dose of AP-7 (0.5 microg/1 microl, P<.05). The frequency of rearing, grooming, and closed arm entries was not affected by the treatment. We conclude that NMDA and non-NMDA-glutamatergic blockade in the Acc lead to a behavioral disinhibition of cortical influences with the median doses, but that at higher doses the blockers have an anxiolytic-like effect.     

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.     

Automatic analysis of immunocytochemically stained tissue samples

An automatic colour image segmentation and cell counting software system has been developed for immunocytochemical analysis of stained tissue samples. The system was designed to count the total number of positive and negative cells in tissue samples treated with cytokine DNA probes from pigs naturally parasitised with Taenia solium metacestodes, using in situ hybridisation. A reaction index was calculated as the ratio of the number of cells with a positive reaction to the total number of cells (positives plus negatives) for each of five different probes. The objectives of automatic counting were to improve the reproducibility of the analysis and reduce the processing time of large image batches. A fast KNN classifier was used for colour segmentation. Watershed segmentation combined with edge detection was used to isolate individual cells that were then automatically labelled, using the results of the corresponding colour segmented image. Validation was performed on 122 non-training digital images with a total of 1069 positive cells and 1459 negative cells, with the following results: a mean true positive rate of 90.2% for positive cells and a mean true positive rate of 85.4% for negative cells. The corresponding mean false positive rates were 9.6% and 6.6%. The mean reaction index error of the automatic analysis was 5.35%. The processing of each digital image took 10 s on a Pentium IV PC.     

Prävalenz des Plasmafaktormangels in der Wiener Bevölkerung

Summary A screening investigation for the presence of risk factors for the development of atherosclerosis demonstrates a plasma factor deficiency in 0,8% in the Viennese population. These findings are in agreement with the data of a newborn screening performed earlier. All the persons were clinically healthy. In 4 of them at least 1 family member suffered from the same defect. The pathogenetic relevance of the plasma factor defect for thrombophilia at young age is discussed.

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Abkürzungsverzeichnis VIP Viennese Initiative for Prostaglandin - PGI2 Prostazyklin - PG Prostaglandin - PF Plasmafaktor - PRP pättchenreiches Plasma - HUS hämolytischurämisches Syndrom - PF4 Plättchenfaktor 4 - TG Thromboglobulin - TXB2 Thromboxan B2 Diese Untersuchung im Rahmen des VIP(VienneseInitiative forProstaglandin)-Screenings wurde vom Medizinisch-Wissenschaftlichen Fonds des Bürgermeisters der Bundeshauptstadt Wien unterstützt     

Gastroesophageal reflux in asthmatic patients: an incidence study and clinical correlation

The prevalence of gastroesophageal reflux (GER) in asthmatic patients is elevated, but the exact frequency remains unknown. The relationship between GER and asthma has not been investigated in Mexico. The objective of this study is to know the frequency of GER in Mexican asthmatic patients and the possible relationship with the severity of asthma. Fifty patients with adult-onset asthma were studied. AII of them fulfill the diagnostic criteria of the National Institutes of Health, U.S. The evaluation included a symptoms questionnaire, spirometry, esophageal manometry, 24-h esophageal pH-recording, and an upper gastrointestinal endoscopy. Twenty-three patients had mild asthma (46%), 16 moderate (32%) and 11 had severe asthma (22%). Twenty-seven (54%) reported heartburn and regurgitation at least twice a week. The esophageal pH-recording showed pathologic GER in 37 subjects (74%) and endoscopic esophagitis was found in 7 cases (14%). The pH-recording showed pathologic GER in 13 patients with mild asthma (57%), in 13 with moderate asthma (81%) and in all patients with severe asthma (100%). The frequency of GER in Mexican asthmatic patients is high and increases proportionately with the severity of asthma. This factor must be considered in the integral evaluation of these patients.     

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells

Insulin induces vasodilatation in human subjects and increases l-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. l-Arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca²⁺, l-arginine and l-citrulline levels, l-[³H]citrulline formation from l-[³H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased l-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca²⁺, l-[³H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the l-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.     

Relative frequency of rotavirus subgroups 1 and 2 in Venezuelan children with gastroenteritis as assayed with monoclonal antibodies.

Monoclonal antibodies recently developed against the 42,000-dalton protein of two rotavirus strains were used in an enzyme-linked immunosorbent assay to determine the subgroup specificity of 252 specimens collected during a 45-month period from Venezuelan children with rotavirus gastroenteritis. Subgroup 2 rotavirus was shed by 85% of the children, whereas only 14% shed subgroup 1 rotavirus (one-half of them in a 3-month period). No differences were found in the occurrence of fever and vomiting between children shedding either rotavirus subgroup, but it appeared that the syndrome tended to last longer in children shedding subgroup 2 rotavirus. The monoclonal subgrouping enzyme-linked immunosorbent assay seemed to be more sensitive than an immune adherence hemagglutination assay, an enzyme-linked immunosorbent assay with polyclonal antibodies, or the electrophoretic analysis of RNA extracted from the virus. Overall, 99% of the specimens could be subgrouped by this assay.     

A meta-analysis of the effect of HIV prevention interventions on the sex behaviors of drug users in the United States

We examined the effectiveness of 33 U.S.-based HIV intervention studies in reducing the sexual risk behaviors of drug users by reducing unprotected sex or increasing the use of male condoms. The studies, identified as of June 1998, through the HIV/AIDS Prevention Research Synthesis project, were published in 1988 or later, measured behavioral or biologic outcomes, used experimental designs or certain quasi-experimental designs, and reported sufficient data for calculating an effect size for sexual risk reduction. Of the 33 studies, 94% recruited injection drug users; 21% recruited crack users. The mean age of participants was 36 years. Almost all studies were randomized (94%), provided another HIV intervention to the comparison groups (91%), and evaluated behavioral interventions (91%). On average, interventions were conducted in 5 sessions (total, 10 hours) during 4.5 months. Interventions compared with no interventions were strong and significant (k = 3; odds ratio [OR], 0.60; 95% confidence interval [CI], 0.43-0.85). Interventions compared with other HIV interventions showed a modest additional benefit (k = 30; OR, 0.91; 95% CI, 0.81-1.03). When we extrapolated our result (an OR of 0.60) to a population with a 72% prevalence of risk behavior, the proportion of drug users who reduced their risk behaviors was 12.6% greater in the intervention groups than in the comparison groups. Our meta-analysis shows that interventions can lead to sexual risk reduction among drug users and justifies providing interventions to drug users. Developing interventions with stronger effects to further reduce sexual risk behaviors among drug users must remain a high priority.     

Biochemical and electron microscopic observations of vaccinia virus morphogenesis in HeLa cells after hydroxyurea reversal

Examination of thin sections of S3 HeLa cells propagated in suspension cultures and infected with vaccinia virus in the presence of hydroxyurea (HU) showed accumulation of immature virus particles. Upon removal of the drug the typical cytoplasmic development of virions was observed, confirming the usefulness of HU for separating early and late events in the replication cycle of poxvirions in spinner cultured cells and for studying viral morphogenesis under quasisynchronous conditions.Six hours after HU reversal, cytoplasm prepared by lysis of cells in Nonidet P-40 was fractionated by sucrose density centrifugation. Particles obtained by the fractionation procedure were negatively stained and their morphology characterized as follows: fraction I contained complete virions and cores with lateral bodies; fraction II contained particles 2300 × 3000 Å in size which resembled viral cores but lacked some of the surface layers found on such subviral particles prepared in vitro; fraction III contained spherical particles 2600–2800 Å in diameter and tubular structures of varying lengths and 1300 Å in diameter; fraction IV yielded particles which morphologically resembled viral cores but had irregular surfaces and appeared damaged; their sedimentation behavior during density gradient centrifugation and subsequent isolation suggested that they may be membrane bound in the cytoplasm and were mechanically released during isolation.When cytoplasmic samples were centrifuged in velocity gradients under conditions where subviral and viral particles were pelleted, progeny DNA was found throughout the gradient. In sucrose gradients prepared in 10^?3M phosphate buffer, pH 7.2, this DNA could be resolved into three classes. A minor fraction (10% or less of the radioactivity) remained at the top of the gradient, major components sedimented at 50–100 S (about 30–40%), the third > 100 S (10–15%). The remainder of the radioactivity was in the pellet. When the 50–100 S fraction was analyzed in alkaline gradients, the denatured DNA sedimented at about 30 S.Early after HU reversal (1–3 hr), before significant assembly of viral DNA into DNase resistant structures had occurred, 85–90% of the newly synthesized viral DNA could be recovered in the form of virosomes.Thin sections of viral DNA protein complexes when examined in the electron microscope were found to be heterogeneous in composition. Irregular ovoid bodies, 0.8–1.0 μm by 0.5 to 0.7 μm in size and filamentous material often in large aggregates composed of fibrils about 20 Å in diameter comprised the major components observed in virosome preparations.     

The effectiveness of a treatment protocol for male lower urinary tract symptoms in general practice: a practical randomised controlled trial

BACKGROUND: Randomised controlled trials have shown the efficacy of several treatment modalities for lower urinary tract symptoms (LUTS) in selected populations. The effectiveness in daily practice has hardly been investigated, especially in primary care and is dependent on choices between all possible treatment options and best investigated in a comprehensive study, including all treatment modalities (watchful waiting, alpha-blockers, 5-alpha-reductase inhibitors, and surgery). AIM: Assessment of the effectiveness of a comprehensive treatment protocol for LUTS in primary care. DESIGN OF STUDY: Randomised controlled trial. SETTING: Fourteen general practices in the Netherlands. METHOD: Intervention: treatment protocol based on a formalised expert opinion. Control condition: usual care. Study population: 208 subjects with moderate to severe LUTS (IPSS > or =8, median = 13). OUTCOME MEASURES: symptom severity (IPSS [International Prostate Symptom Score]), bother score (Dan-PSS [Danish Prostate Symptom Score]), and maximum urinary flow (Q(max)); incidence of acute urinary retention and urinary tract infections. RESULTS: In the intervention group markedly more subjects used an alpha-blocker at end of follow-up than in the usual care group (24% versus 6%). No significant differences were found between intervention and control group in IPSS, Q(max) or Dan-PSS. CONCLUSION: alpha-blockers and watchful waiting are the most frequent treatment modalities for LUTS in primary care. Our study showed no evidence that a protocol using well-defined indications for all possible treatment modalities based on a formalised expert opinion procedure has added value. Based on our results, we cannot recommend a broadening of the indication for alpha-blockers, which, however, seems to be the current trend.