Reduced HIV-1 infectability of CD4+ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines

Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A-K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.     

Nuclear migration, nucleokinesis and lissencephaly

During the past 20 years, biologists have become used to finding that proteins first identified in simple, genetically manipulable eukaryotic organisms are conserved in higher eukaryotes. This article draws attention to the similarity between NUDF protein, which is required for nuclear migration in the filamentous fungus Aspergillus nidulans, and a mammalian homologue, LIS1, whose malfunction causes lissencephaly, a neuronal migration disease. The authors suggest that there might be an underlying similarity of mechanism between nuclear migration in the fungus and neuronal migration in the brain.     

Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system?

Despite a large body of evidence showing the beneficial effects of successful treatment of anemia with recombinant human erythropoietin (EPO) in patients with end-stage renal disease, controversy remains as to whether EPO treatment of anemia can improve the nutritional status in patients on maintenance hemodialysis. This prompted us to conduct a prospective study in 41 hemodialysis patients with basal hemoglobin less than 9 g/dl. The dose of EPO was increased for 12 weeks to achieve the target hemoglobin concentration of 10 g/dl and then titrated in the following 12 weeks to maintain the target value. Nutritional status was assessed at baseline and after 6 months of follow-up, using the global protein-calorie malnutrition (PCM) index proposed by Bilbrey and Cohen. A low global PCM score indicates better nutrition. The results showed that hemoglobin values significantly increased from 8.7 +/- 0.8 g/dl at baseline to 10.7 +/- 0.5 g/dl in the 6th month (p < 0.001). No significant changes were observed in the normalized protein catabolic rate and Kt/V during the study period. Global PCM scores improved from 30.0 +/- 7.5 to 23.6 +/- 3.1 (p < 0.001) and paralleled the correction of anemia by EPO treatment. The data were consistent with a major improvement in the nutritional markers of relative body weight, triceps skinfold, midarm circumference, midarm muscle circumference, serum albumin, serum transferrin and total lymphocyte count in the 6th month as compared to baseline. The percentages of mild and moderate-severe PCM at baseline were 32 and 58%, respectively. These percentages were significantly reduced during the 6th month to 20 and 30%, respectively (p = 0.0004). In summary, correction of renal anemia with EPO improves the nutritional status in hemodialysis patients. A postulated mechanism is that EPO may exhibit anabolic effects, with a better utilization of ingested protein.     

A secondary prevention trial of antioxidant vitamins and cardiovascular disease in women. Rationale, design, and methods. The WACS Research Group

The evidence for a potential benefit of antioxidant vitamins in the prevention and therapy of atherosclerotic disease is derived from laboratory, clinical, and observational epidemiologic studies but remains inconclusive. Data from randomized clinical trials are sparse, particularly for women. Therefore, it is both timely and important to conduct large-scale primary and secondary prevention trials of antioxidants and cardiovascular disease (CVD). The Women's Antioxidant and Cardiovascular Study (WACS) is a randomized, double-blind, placebo-controlled secondary prevention trial of the balance of benefits and risks of antioxidant vitamins (vitamins E and C, and beta-carotene) among 8000 women with preexisting CVD. This secondary prevention trial will be conducted as a companion to the recently started Women's Health Study, a primary prevention trial of vitamin E and beta-carotene, as well as aspirin. In the WACS, US female health professionals aged 40 years and older with a history of myocardial infarction, angina pectoris, coronary revascularization, stroke, transient cerebral ischemia, carotid endarterectomy, or peripheral artery surgery will be randomly assigned, utilizing a 2 x 2 x 2 factorial design, to receive vitamin E, vitamin C, beta-carotene, and/or placebo. Cardiovascular end points include nonfatal myocardial infarction, nonfatal stroke, coronary revascularization procedures, and total CVD mortality. The present article describes the rationale, design, and methods of the trial.     

Reversal of CPT-11 resistance of lung cancer cells by adenovirus-mediated gene transfer of the human carboxylesterase cDNA

To evaluate the concept that transfer of the human carboxylesterase (CE) gene will overcome the drug resistance of a solid tumor to CPT-11 (irinotecan), we used an adenovirus vector (AdCMV.CE) carrying human CE cDNA to infect CPT-11-resistant A549 human adenocarcinoma cells (A549/CPT) in vitro and in vivo and evaluated cell growth over time. The A549/CPT cells, selected by stepwise and continuous exposure of parental A549 cells to CPT-11 over 10 months, had a 6-fold resistance to CPT-11 and 42% CE activity in comparison with parental A549 cells. AdCMV.CE infection resulted in an increase in functional CE protein in resistant cells in vitro that was sufficient to convert CPT-11 to its active metabolite, SN-38, and effectively suppressed resistant cell growth in vitro in the presence of CPT-11. When AdCMV.CE was directly injected into established s.c. resistant A549-based tumors in nude mice receiving CPT-11, there was a 1.8-fold reduction in tumor size at day 20 compared to that of controls (P < 0.05). These observations suggest that adenovirus-mediated gene transfer of the human CE gene and concomitant administration of CPT-11 may have potential as a strategy for local control of acquired CPT-11 resistance of solid tumors.     

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.