Crystallization and crystal packing of Proteus mirabilis PR catalase

The tetrameric catalase from Proteus mirabilis PR (EC 1.11.1.6), known to bind NADPH, has been crystallized by the hanging-drop method in a form apparently depleted in dinucleotide. The crystals belong to the hexagonal space group P6(2)22 with a = b = 111.7 A, c = 248.8 A. There is one subunit in the asymmetric unit. Data were collected to 2.9 A at the L.U.R.E. (Orsay) synchrotron radiation facility. The tetramers have been located in the crystal, centered on the site (1/2, 0, 0) with 222 symmetry.     

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory β-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the β-subunit. An interaction between ZnT-1 and the β-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming α₁-subunit of the LTCC. Similarly, a decrease in the surface expression of the α₁-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC β-subunit and that it involves a decrease in the trafficking of the LTCC α₁-subunit to the surface membrane.     

First Reported Case of Cryptococcus gattii in the Southeastern USA: Implications for Travel-Associated Acquisition of an Emerging Pathogen

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient''s travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.     

Nonvalvular atrial fibrillation: evidence for a prothrombotic state

OBJECTIVE: To determine whether patients with nonvalvular atrial fibrillation (NVAF) have prothrombotic changes compared with patients in sinus rhythm. DESIGN: Cross-sectional study. Hemostatic function compared in NVAF patients without prior embolic event (transient ischemic attack or embolic stroke) and control subjects without prior thrombotic stroke, and in NVAF patients with prior embolic event and control subjects with prior thrombotic stroke. SETTING: Internal medicine outpatient group practice and anticoagulation clinic in 2 teaching hospitals. PATIENTS: A total of 75 NVAF patients (50 without and 25 with prior embolic event) and 42 control patients (31 without and 11 with prior thrombotic stroke) recruited concurrently over 18 months during 1990-91. OUTCOME MEASURES: Platelet count, prothrombin time (PT), partial thromboplastin time (PTT), and plasma levels of hemoglobin, fibrinogen, von Willebrand factor antigen, factor VIII, fibrin D-dimer, antithrombin III, protein C, protein S, fibrinopeptide A and prothrombin fragment F1+2. All statistical analyses were performed after adjustments for age and sex. RESULTS: The NVAF patients without a prior embolic event had significantly higher mean hemoglobin and fibrinogen levels (p < 0.001 and p = 0.05, respectively) than the control subjects without prior thrombotic stroke. The 29 NVAF patients not taking warfarin (none had had an embolic event) had significantly lower mean protein C and protein S levels (p = 0.012 and p < 0.001, respectively) and a significantly higher fibrinopeptide A level (p = 0.03, after exclusion of outliers) than the control subjects without prior stroke. The NVAF patients with a prior embolic event had alterations in the hemostatic variables similar to those seen in the control patients with a prior thrombotic stroke. The latter had significantly higher fibrinogen, von Willebrand factor antigen and factor VIII levels (p = 0.04, 0.002 and 0.002, respectively) and significantly lower protein S levels (p = 0.02) than the control subjects without prior stroke. CONCLUSIONS: NVAF patients without a history of an embolic event show evidence of a prothrombotic state compared with patients in sinus rhythm who have not had a thrombotic stroke. NVAF patients with a history of an embolic event show evidence of a prothrombotic state similar to that of patients in sinus rhythm who have had a thrombotic stroke. Prospective studies are needed to determine whether these abnormalities predict higher risk of stroke in individual NVAF patients.     

Vascular fluid shifts and endocrine responses to exercise in the heat. Effect of rehydration

This study examines the relationships between vascular changes and endocrine responses to prolonged exercise in the heat, associated with dehydration and rehydration by fluids of different osmolarity. Five subjects were exposed, in a 34 degrees C environment for 4 h of intermittent exercise on a cycle ergometer at 85 +/- 12 Watts (SD). Fluid regulatory hormones and cortisol were analysed in 3 experimental sessions: one without any fluid supplement (NO FLUID), and two with progressive rehydration, either by spring water (WATER) or isotonic solution (ISO), given after 70 min of exercise. Results were expressed in terms of differences between the mean values observed at the end of the exercise and the first hour values taken as references. Dehydration (NO FLUID) elicited a 4.0 +/- 0.8% (SE) decrease in plasma volume (PV) and an increase in osmolarity (8.4 +/- 3.1 mosmol X l-1). Concomitantly, plasma aldosterone (PA), renin activity (PRA), arginin vasopressin (AVP) and cortisol (PC) levels increased greatly in response to exercise in the heat (PA: 37.2 +/- 10.8 ng. 100 ml-1; PRA: 13.4 +/- 2.5 ng X ml-1 X h-1; AVP: 3.8 +/- 1.3 pg X ml-1; PC: 12.2 +/- 2.7 micrograms X 100 ml-1). Rehydration with water led to decreased osmolarity (-8.2 +/- 2.1 mosmol X l-1) with no significant changes in PV. With ISO, PV increased by 6.0 +/- 1.3% and the decrease in osmolarity was-5.8 +/- 1.8 mosmol X l-1. With both modes of rehydration, the increases in PRA, AVP and cortisol were blunted; only ISO prevented the rise in PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity.

The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the B?yum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies.