The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation ofI _Cl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (–)-indolactam did not affectI _Cl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19–31 pseudosubstrate peptide, did not influenceI _Cl,vol. Trifluoperazine and tamoxifen fully blockedI _cCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca²⁺] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 ol/1) and genistein (100 ol/1) did not affectI _Cl,vol Exposing CPAE cells to lysophosphatidic acid (1mol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125^FAK in endothelial cells, neither evoked a Cl^– current nor affectedI _Cl,vol Neither wortmannin (10 mol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affectedI _Cl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl^– current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl^– current induced by hypotonicity.     

Clinical and cost-effectiveness of a new nurse-led continence service: a randomised controlled trial

BACKGROUND: Continence services in the UK have developed at different rates within differing care models, resulting in scattered and inconsistent services. Consequently, questions remain about the most cost-effective method of delivering these services. AIM: To evaluate the impact of a new service led by a continence nurse practitioner compared with existing primary/secondary care provision for people with urinary incontinence and storage symptoms. DESIGN OF STUDY: Randomised controlled trial with a 3- and 6-month follow-up in men and women (n = 3746) aged 40 years and over living in private households (intervention [n = 2958]; control [n = 788]). SETTING: Leicestershire and Rutland, UK. METHOD: The continence nurse practitioner intervention comprised a continence service provided by specially trained nurses delivering evidence-based interventions using predetermined care pathways. They delivered an 8-week primary intervention package that included advice on diet and fluids; bladder training; pelvic floor awareness and lifestyle advice. The standard care arm comprised access to existing primary care including GP and continence advisory services in the area. Outcome measures were recorded at 3 and 6 months post-randomisation. RESULTS: The percentage of individuals who improved (with at least one symptom alleviated) at 3 months was 59% in the intervention group compared with 48% in the standard care group (difference of 11%, 95% CI = 7 to 16; P<0.001) The percentage of people reporting no symptoms or 'cured' was 25% in the intervention group and 15% in the standard care group (difference of 10%, 95% CI = 6 to 13, P = 0.001). At 6 months the difference was maintained. There was a significant difference in impact scores between the two groups at 3 and 6 months. CONCLUSIONS: The continence nurse practitioner-led intervention reduced the symptoms of incontinence, frequency, urgency and nocturia at 3 and 6 months; impact was reduced; and satisfaction with the new service was high.     

Order of genetic events is critical determinant of aberrations in chromosome count and structure

A sequential acquisition of genetic events is critical in tumorigenesis. A key step is the attainment of infinite proliferative potential. Acquisition of this immortalization requires the activation of telomerase in addition to other activities, including inactivation of TP53 and the retinoblastoma family of tumor-suppressor proteins. However, the importance of the order in which these genetic events occur has not been established. To address this question, we used a panel of normal mammary fibroblasts and endothelial cultures that were immortalized after transduction with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive mutant of the SV40 large-tumor (tsLT) oncoprotein in different orders in early- and late-passage stocks. These lines were maintained in continuous culture for up to 90 passages, equivalent to >300 population doublings (PDs) post-explantation during 3 years of continuous propagation. We karyotyped the cultures at different passages. Cultures that received hTERT first followed by tsLT maintained a near-diploid karyotype for more than 150 PDs. However, in late-passage stocks (>200 PDs), metaphase cells were mostly aneuploid. In contrast, the reverse order of gene transduction resulted in a marked early aneuploidy and chromosomal instability, already visible after 50 PDs. These results suggest that the order of genetic mutations is a critical determinant of chromosome count and structural aberration events.     

Connective tissue growth factor expression and Smad signaling during mouse heart development and myocardial infarction.

Connective tissue growth factor (CTGF) is reported to be a target gene of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) in vitro. Its physiological role in angiogenesis and skeletogenesis during mouse development has been described recently. Here, we have mapped expression of CTGF mRNA during mouse heart development, postnatal adult life, and after experimental myocardial infarction. Furthermore, we investigated the relationship between CTGF and the BMP/TGFbeta signaling pathway in particular during heart development in mutant mice. Postnatally, CTGF expression in the heart became restricted to the atrium. Strikingly, 1 week after myocardial infarction, when myocytes have disappeared from the infarct zone, CTGF and TGFbeta expression as well as activated forms of TGFbeta but not BMP, Smad effector proteins are colocalized exclusively in the fibroblasts of the scar tissue, suggesting possible cooperation between CTGF and TGFbeta during the pathological fibrotic response.     

Different strains of Mycobacterium tuberculosis cause various spectrums of disease in the rabbit model of tuberculosis

The rabbit model of tuberculosis has been used historically to differentiate between Mycobacterium tuberculosis and Mycobacterium bovis based on their relative virulence in this animal host. M. tuberculosis infection in market rabbits is cleared over time, whereas infection with M. bovis results in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits to M. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of the M. tuberculosis Erdman strain were required than of M. tuberculosis H37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only with M. bovis infection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences between M. tuberculosis strains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specific M. tuberculosis genes important in human disease.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.     

Genetic association between the phospholipase A2 gene and unipolar affective disorder: a multicentre case-control study

The co-segregation in one pedigree of bipolar affective disorder with Darier's disease whose gene is on chromosome 12q23-q24.1, and findings from linkage and association studies with the neighbouring gene of phospholipase A2 (PLA2) indicate that PLA2 may be considered as a candidate gene for affective disorders. All relevant genetic association studies, however, were conducted on bipolar patients. In the present study, the possible association between the PLA2 gene and unipolar affective disorder was examined on 321 unipolar patients and 604 controls (all personally interviewed), recruited from six countries (Belgium, Bulgaria, Croatia, Germany, Greece, and Italy) participating in the European Collaborative Project on Affective Disorders. After controlling for population group and gender, one of the eight alleles of the investigated marker (allele 7) was found to be more frequent among unipolar patients with more than three major depressive episodes than among controls (P<0.01); genotypic association was also observed, under the dominant model of genetic transmission (P<0.02). In addition, presence of allele 7 was correlated with a higher frequency of depressive episodes (P<0.02). These findings suggest that structural variations at the PLA2 gene or the chromosomal region around it may confer susceptibility for unipolar affective disorder.     

Phosphatidylcholine-Specific Phospholipase C from Listeria monocytogenes Is an Important Virulence Factor in Murine Cerebral Listeriosis

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (ΔinlAB2)- and a PlcB (ΔplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384–2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the ΔplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the ΔinlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the ΔplcB2 strain compared to that of mice infected with the WT or ΔinlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the ΔplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.     

DFNB40, a recessive form of sensorineural hearing loss, maps to chromosome 22q11.21-12.1

We report on a novel localization for a recessive form of deafness (DFNB), by linkage analysis in an Iranian consanguineous family. Affected individuals suffer from prelingual profound sensorineural hearing loss. Genome-wide analysis led to the characterization of a new locus, DFNB40, which maps to an approximately 9 Mb interval between markers D22S427 and D22S1144 at chromosome 22q11.21-12.1. Maximum lod score of 3.09 was obtained with D22S1174. Since the Bronx waltzer (bv) mouse mutant, characterized by waltzing behavior, deafness, and degeneration of cochlear inner hair cells, has been mapped to the syntenic region on murine chromosome 5, we suggest that DFNB40 and bv may result from orthologous gene defects.