EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

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Dendritic cell-mediated viral transfer to T cells is required for human immunodeficiency virus type 1 persistence in the face of rapid cell turnover

Human immunodeficiency virus type 1 (HIV-1)-infected and activated CD4(+) T cells have short half-lives in vivo (<2 days). We have established an in vitro culture system in which infected T cells are turned over frequently to provide a model system that examines this important facet of in vivo HIV-1 replication. We observed that virus replication in T cells under rapid-turnover conditions was possible only when immature dendritic cells or DC-SIGN-expressing cells mediated HIV-1 transmission to T cells. Virus replication was initiated more rapidly in T cells infected with the cell-associated form of virus compared to infection by the cell-free route. This accelerated transfer of virus required adhesion molecule-mediated interactions between the virus-presenting cell and T cell, but surprisingly, HIV-1 transfer could occur independently of DC-SIGN (DC-specific intracellular adhesion molecule 3 [ICAM-3]-grabbing nonintegrin)in the dendritic-cell-T-cell cocultures. These results suggest that dendritic cell-mediated transmission of HIV-1 enables virus replication under conditions of rapid cell turnover in vivo.     

Singlet oxygen: a potential culprit in myocardial injury?

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca ²⁺ uptake; from 2.27 ± 0.05 to 0.62 ± 0.05 µmol Ca⁺/mg.min (mean ± SE) (P < 0.01) and Ca²⁺-ATPase activity from 2.08 ± 0.05 µmol Pi/min. mg to 0.28 ± 0.04 µmol Pi/min. mg (mean ± SE) (P < 0.01). The inhibition of calcium uptake and Ca²⁺-ATPase activity by rose bengal derived activatedoxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca²⁺-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca²⁺-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H₂O₂ + Fe²⁺ -EDTA) as well as H₂O₂ (12 mM) were without any effect on the 97,000 dalton Ca²⁺-ATPase band ofsarcoplasmic reticulum. The results suggest that oxidative damage of cardiac sarcoplasmic reticulum may be mediated by singlet oxygen. This may represent an important mechanism by which the oxidative injury to the myocardium induces both a loss of tension development and arrhythmogenesis.     

Involvement of polycomb-group genes in establishing HoxD temporal colinearity

Temporal colinearity in mouse HoxD is dependent on repressive activity of sequences within the 5' end of the complex. We show that a 5-kb DNA fragment from this region represses transgenes when combined in mouse as well as in Drosophila melanogaster. Moreover, repressive activity in Drosophila depends on some members of the Polycomb-group (PcG) genes, for example, extra sex combs. We also showed direct association of these factors with the repressive fragment, both in transgenic flies and in the context of the native mouse HoxD complex. These results suggest that the global repressive region of the HoxD complex functions in two very different species and that some PcG genes are involved in establishing the early repressive state of the HoxD complex, thus contributing to temporal colinearity.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

Protective effect of curcumin against intracerebral streptozotocin induced impairment in memory and cerebral blood flow

AimsThe aim of the present study is to investigate the effect of curcumin on cerebral blood flow (CBF), memory impairment, oxidative stress and cholinergic dysfunction in intracerebral (IC) streptozotocin (STZ) induced memory impairment in mice.Main methodsMemory impairment was induced by STZ (0.5 mg/kg, IC) administered twice with an interval of 48 h in mice. Memory function was assessed by Morris water maze and passive avoidance test. CBF was measured by Laser Doppler Flowmetry (LDF). To study the preventive effect, curcumin (10, 20 and 50 mg/kg, PO) was administered for 21 days starting from the first dose of STZ. In another set of experiment, curcumin was administered for 7 days from 19th day after confirming STZ induced dementia to observe its therapeutic effect. Biochemical parameters of oxidative stress and cholinergic function were estimated in brain on day 21.Key findingsThe major finding of this study is that STZ (IC) caused a significant reduction in CBF along with memory impairment, cholinergic dysfunction and enhanced oxidative stress. Curcumin dose dependently improved CBF in STZ treated mice together with amelioration of memory impairment both in preventive and therapeutic manner.SignificanceThe present study clearly demonstrates the beneficial effects of curcumin, the dietary staple of India, on CBF, memory and oxidative stress which can be exploited for dementia associated with age related vascular and neurodegenerative disorders.     

The mitotic checkpoint gene BubR1 has two distinct functions in mitosis

BubR1 is one of two putative vertebrate homologs of the yeast spindle checkpoint protein Bub1. We have used deletion and point mutants to elucidate the functions of BubR1 in mitosis. The nocodazole-activated spindle checkpoint of HeLa cells was disrupted by expression of a 39 amino acid fragment (residues 382-420) of BubR1 containing the Bub3-binding GLEBS motif. In contrast, we observed normal checkpoint function in a truncation mutant comprising residues 1-477, despite the lack of the C-terminal BubR1 kinase domain. In the absence of nocodazole, expression of the 477 amino acid fragment slowed progress through prometaphase of mitosis, causing accumulation of mitotic cells. This accumulation was also seen in a kinase dead mutant. The prolongation of mitosis required both kinetochore binding and an intact, functional spindle checkpoint. The prolongation of mitosis by kinase deficient BubR1 constructs indicates a crucial role for the BubR1 C-terminal kinase domain in chromosome movement, in addition to the role of the N-terminus in the checkpoint.     

Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.