Oscillatory membrane currents paradoxically induced via NO-activated pathways in detrusor cells

Oscillatory inward membrane currents (I(oscil-in)) reflecting intracellular Ca(2+) ([Ca(2+)](i)) activity in detrusor cells, are thought to play an important role in producing tonic bladder contractions during micturition. The present patch clamp study revealed a new activation mechanism: sodium nitroprusside (SNP), a nitric oxide (NO) donor induced I(oscil-in) in a subpopulation of detrusor cells. The inhibitory effect of niflumic acid on SNP-induced I(oscil-in) suggests that Ca(2+)-activated Cl(-) channels are responsible for this current. In addition, SNP-induced I(oscil-in) required the cooperation of Ca(2+) influx through SK&F96365-sensitive channels and intracellular Ca(2+) release channels sensitive to ryanodine but insensitive to xestospongin C (XeC). This is also true for muscarinic agonist (carbachol: CCh)-induced I(oscil-in). However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, suppressed SNP-induced I(oscil-in) but not CCh-induced I(oscil-in). The results suggest that a subpopulation of detrusor cells employ the NO/cGMP cascade to potentiate bladder contraction. Mechanisms underlying NO-induced I(oscil-in) are likely to contribute not only to the physiology but also to the pathophysiology of the lower urinary tract.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Diminished proliferation of human immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-2 (IL-2) production and is recovered by exogenous IL-2

Virus-specific CD4(+) T-cell function is thought to play a central role in induction and maintenance of effective CD8(+) T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4(+) T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4(+) T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-gamma in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer(+) or total-Gag-specific CD4(+) T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4(+) T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4(+) T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.     

Molecular architecture of a eukaryotic DNA transposase

Mobile elements and their inactive remnants account for large proportions of most eukaryotic genomes, where they have had central roles in genome evolution. Over 50 years ago, McClintock reported a form of stress-induced genome instability in maize in which discrete DNA segments move between chromosomal locations. Our current mechanistic understanding of enzymes catalyzing transposition is largely limited to prokaryotic transposases. The Hermes transposon from the housefly is part of the eukaryotic hAT superfamily that includes hobo from Drosophila, McClintock's maize Activator and Tam3 from snapdragon. We report here the three-dimensional structure of a functionally active form of the transposase from Hermes at 2.1-A resolution. The Hermes protein has some structural features of prokaryotic transposases, including a domain with a retroviral integrase fold. However, this domain is disrupted by the insertion of an additional domain. Finally, transposition is observed only when Hermes assembles into a hexamer.     

PEX14 binding to Arabidopsis PEX5 has differential effects on PTS1 and PTS2 cargo occupancy of the receptor

PEX5 acts as a cycling receptor for import of PTS1 proteins into peroxisomes and as a co-receptor for PEX7, the PTS2 receptor, but the mechanism of cargo unloading has remained obscure. Using recombinant protein domains we show PEX5 binding to the PEX14N-terminal domain (PEX14N) has no effect on the affinity of PEX5 for a PTS1 containing peptide. PEX5 can form a complex containing both recombinant PTS1 cargo and endogenous PEX7-thiolase simultaneously but isolation of the complex via the PEX14 construct resulted in an absence of thiolase, suggesting a possible role for PEX14 in the unloading of PTS2 cargos.     

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing,Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.