Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Gene expression profiles in murine hematopoietic stem cells revisited: Analysis of cDNA libraries reveals high levels of translational and metabolic activities

Gene expression studies from hematopoietic stem cell (HSC) populations purified to variable degrees have defined a set of sternness genes. Unexpectedly, results also hinted toward a HSC chromatin poised in a wide-open state. With the aim of providing a robust tool for further studies into the molecular biology of HSCs, the studies herein describe the construction and comparative molecular analysis of A-phage cDNA libraries from highly purified HSCs that retained their long-term repopulating activities (long-term HSCs [LT-HSCs]) and from short-term repopulating HSCs that were largely depleted of these activities. Microarray analysis of the libraries confirmed the previous results but also revealed an unforeseen preferential expression of translation- and metabolism-associated genes in the LT-HSCs. Therefore, these data indicate that HSCs are quiescent only in regard of proliferative activities but are in a state of readiness to provide the metabolic and translational activities required after induction of proliferation and exit from the HSC pool.

Outcomes after stroke: Basic and instrumental activities of daily living, community reintegration and generic health status

This study describes the discharge destination, basic and instrumental activities of daily living (ADL), community reintegration and generic health status of people after stroke, and explored whether sociodemographic and clinical characteristics were associated with these outcomes. Participants were 51 people, with an initial stroke, admitted to an acute hospital and discharged to the community. Admission and discharge data were obtained by chart review. Follow-up status was determined by telephone interview using the Modified Barthel Index, the Assessment of Living Skills and Resources, the Reintegration to Normal Living Index, and the Short-Form Health Survey (SF-36). At follow up, 57% of participants were independent in basic ADL, 84% had a low risk of experiencing instrumental ADL difficulties, most had few concerns with community reintegration, and SF-36 physical functioning and vitality scores were lower than normative values. At follow up, poorer discharge basic ADL status was associated with poorer instrumental ADL and community reintegration status, and older participants had poorer instrumental ADL, community reintegration and physical functioning. Occupational therapists need to consider these outcomes when planning inpatient and post-discharge intervention for people after stroke.

A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors

Purpose: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study. Experimental Design: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2). Results: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C-max correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure, No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >= 6 months. Conclusion: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.