Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop

The chemokine receptor CCR5 acts as an essential cofactor for cell entry by macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains, whereas CXCR4 acts as an essential cofactor for T-cell-line-adapted strains. We demonstrated that the specific amino acids in the V3 loop of the HIV-1 envelope protein that determine cellular tropism also regulate chemokine coreceptor preference for cell entry by the virus. Further, a strong correlation was found between HIV-1 strains classified as syncytium inducing in standard assays and those using CXCR4 as a coreceptor. These data support the hypothesis that progressive adaptation to additional coreceptors is a key molecular basis for HIV-1 phenotypic evolution in vivo.     

A superconducting X-ray spectrometer with a tantalum absorber andlateral trapping

We report the development of Nb-Ta-Al-Al₂O₃-Al superconducting tunnel junction structures for high energy resolution and high efficiency X-ray detection. These devices utilize a Ta X-ray absorber with superconductor “bandgap engineered” quasiparticle trapping to improve charge collection. Experimental results at 0.3 K are presented, showing energy resolution of 102 eV full-width-half-maximum for 6 keV X-rays. Collected charge is in excess of 5×10⁶ electrons. The absorption efficiency is better than 35%. Devices thermally cycle with no change in characteristics     

Insulin propeptides in conditions associated with insulin resistance in humans and their relevance to insulin measurements

Routine insulin assays measure not only biologically active insulin but also the relatively inactive propeptides, proinsulin and desdipeptide proinsulin. Such measurements may be misleading if insulin propeptide levels are increased, as has been reported in patients with non-insulin-dependent diabetes mellitus (NIDDM). Inferences regarding insulin resistance, based on hyperinsulinemia, could thus be invalidated where routine insulin assays have been used. We have measured plasma insulin levels using a routine assay, together with measurements of the major circulating insulin propeptides, intact proinsulin and des 31,32proinsulin, in various clinical situations associated with apparently increased insulin levels and insulin resistance. Major increases of insulin propeptide levels relative to insulin levels were not seen in obese subjects or in patients taking oral contraceptives or danazol, or in obese subjects compared with non-obese controls. Although the insulinemic responses observed with routine radioimmunoassay in these situations associated with insulin resistance are not confounded by major changes in the proportion of circulating insulin propeptides, further studies will be necessary to validate investigations in other insulin-resistant states.     

Phytohemagglutinin-induced gut hyperplasia and the growth of a mouse lymphosarcoma tumor

OBJECTIVE: To identify possible causes for the increased cardiovascular morbidity and mortality seen in patients with primary hyperparathyroidism. DESIGN: Prospective, blind study. SETTING: University hospital, Sweden. SUBJECTS: 44 Patients with primary hyperparathyroidism and 23 (sex and age matched) control patients with atoxic nodular goitres. INTERVENTIONS: Exploration of the neck with removal of pathological parathyroid glands or thyroid resection. Echocardiography before, and one year after, the operation. MAIN OUTCOME MEASURES: Blood pressure and echocardiographic findings. RESULTS: Hyperparathyroid patients had higher blood pressure and greater left atrial diameter than control patients preoperatively. They also had a significantly lower E:A ratio (mitral flow velocity pattern) than the controls (p = 0.02) indicating a disturbance in early diastolic filling of the left ventricle. The E:A ratio correlated negatively with the systolic blood pressure. 19 of the hyperparathyroid patients (43%) had cardiac calcifications as did 14 (61%) of the controls. Most of calcifications were located in the aortic and mitral valves; only a few patients had calcifications in the myocardium. No significant changes had occurred one year after parathyroidectomy, except for a reduction in systolic blood pressure, in the hyperparathyroid patients. CONCLUSION: Echocardiographic investigation of patients with primary hyperparathyroidism shows early signs of left ventricular dysfunction that may be of clinical importance.     

Investigation of I1-imidazoline receptors using microphysiometry and molecular modelling

A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.     

Molecular motion of small molecules in cellulose gels studied by NMR

Measurements of relaxation times, T₁ and T₂, and self-diffusion coefficients, D, for small molecules, viz., H₂O, dioxane and t-butanol, in the gel system cellulose/H₂O are reported and compared with those for H₂O in a polyacrylamide gel of the same polymer content. The temperature dependence of T₁ and D can for all the penetrants be represented by Arrhenius type relations which merely are parallel shifts by the same amount, towards smaller values, of those obtained without polymer. The T₂ values for H₂O in both gels pass through a shallow minimum over the considered temperature interval (14°?44°C). Furthermore, relaxation times T₁ of D₂O in the gel system cellulose/D₂O are reported; in a plot of ln T₁ vs. 1/T, a plateau region is observed at higher temperatures.     

Complete relaxation and conformational exchange matrix (CORCEMA) analysis of NOESY spectra of interacting systems; two-dimensional transferred NOESY

A very general procedure entitled complete relaxation and conformational exchange matrix (CORCEMA) analysis has been developed to analyze the 2D-NOESY spectra of interacting systems undergoing multistate conformational exchange. This is an extension of earlier work from this laboratory on the methodological treatment of multistate conformational exchange [Krishna et al., Biopolymers 19, 2003 (1980)] and the theory of transferred NOESY for finite exchange off-rates [Lee and Krishna, J. Magn. Reson. 98, 36 (1992)]. The current theory is based on generalized rate matrices for relaxation and conformational exchange. The CORCEMA algorithm explicitly incorporates intermolecular dipolar cross relaxation between the molecules when they are complexed. It permits an analysis of NOESY intensities for the intra- as well as intermolecular contacts between the interacting molecules under a variety of binding conditions. Its application is illustrated on two examples of transferred NOESY simulations: (1) a two-state system involving a ligand and an enzyme forming a ligand-enzyme complex, and (2) a three-state system in which the ligand-enzyme complex can undergo a conformational transition from an "open state" to a "closed state," and can include conformational changes in both the complexed ligand and the complexed enzyme, such as hinge-bending motions. Simplifying expressions for generalized matrix analyses are derived for three limiting cases of the three-state system. This three-state example is illustrated using a hypothetical model of the hinge-bending motion in a thermolysin-inhibitor complex. It is shown that: (1) The neglect of cross relaxation between the interacting species in their complexed forms can lead to misleading conclusions on the "bound" conformation of the ligand. (2) If protein-mediated spin diffusion is dominant, caution is needed in analyses based on initial slopes alone due to one's inability to identify the exact range of the initial growth curve under poor signal/noise situations. (3) The neglect of conformational changes upon complexation, e.g., hinge-bending motions of the ligand-enzyme complex, can lead to erroneous results on the nature of "bound" conformations of the ligand. In this case, attempts to analyze the transferred NOESY data with a two-state model will result in a "virtual" conformation for the bound ligand. (4) When the hinge-bending rate is slower than the cross relaxation and enzyme off-rates, the bound conformation of a ligand deduced from the transferred NOESY experiment is more likely to represent nonspecific or weak binding in an open state of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)