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.     

Vaccination against tick-borne encephalitis virus, a flavivirus, prevents disease but not infection, although viremia is undetectable

By adoptive transfer of sera or immunoglobulin preparations, vaccine-induced protection against TBEV has been demonstrated to be mediated by antibodies to the surface protein of TBEV, glycoprotein E. Nevertheless, the mechanism of vaccine-induced protection against TBEV remains unclear. Protection by E antibodies without in vitro neutralization was shown by one group, whereas others found a correlation between protection in vivo and neutralization in vitro. Here, the authors confirm in a mouse model of tick-borne encephalitis (TBE) that immunization with the whole-killed virus vaccine protects mice against a subsequent challenge with a highly lethal dose of virus, i.e. 250 LD50 doses. Vaccine-induced immunity, however, is not completely neutralizing as demonstrated by the development of immune responses to a non-structural virus protein absent from the vaccine, yet expressed in the course of virus replication. Antibodies specific for the non-structural protein 1 (NS1) and cytotoxic T-cells could be detected after, but not prior to, virus challenge of vaccinated animals, establishing that protection by this highly effective vaccine is not equivalent with complete neutralization of the challenge virus.     

Central adiposity and hemodynamic functioning at rest and during stress in adolescents

OBJECTIVE: To examine the impact of central adiposity upon hemodynamic functioning at rest and during stress in adolescents. DESIGN: Cross-sectional, correlational study. SUBJECTS: 46 White and 49 Black normotensive adolescents with family histories of essential hypertension. MEASUREMENTS: Systolic and diastolic blood pressure (SBP, DBP), cardiac output and total peripheral resistance responses were assessed at rest, during postural change, video game challenge and forehead cold stimulation. Specific lower and higher waist-to-hip ratio (WHR) tertiles were created for each gender and then integrated for analyses. This resulted in a lower WHR tertile of 11 Whites and 21 Blacks and an upper WHR tertile of 15 Whites and 17 Blacks. RESULTS: No differences in age, gender or ethnicity proportions were found between tertile groups (all P > 0.21). The upper WHR group showed greater body weight, waist and hip circumferences, body mass index (BMI), triceps skinfold and body surface area (all P < 0.001). Controlling for peripheral (that is, triceps skinfold) and overall (that is, BMI) adiposity, the upper WHR group exhibited greater SBP (that is, peak response minus mean pre-stressor level) to all three stressors and greater DBP reactivity to postural change and cold pressor (all P < 0.05). CONCLUSION: Central adiposity appears to adversely influence hemodynamic functioning during adolescence. Underlying mechanisms responsible for these associations require exploration.     

Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes

Abnormal intracellular Ca2+ handling in hypertrophied and failing hearts is partly due to changes in Ca2+ transporter gene expression, but the mechanisms responsible for these alterations remain largely unknown. We previously showed that intrinsic mechanical load (i.e. spontaneous contractile activity) induced myocyte hypertrophy, and down-regulated SR Ca2+ ATPase (SERCA2) gene expression in cultured neonatal rat ventricular myocytes (NRVM). In the present study, we examined whether extrinsic mechanical load (i.e. cyclic stretch) also induced NRVM hypertrophy, and led to down-regulation of SERCA2 and other Ca2+ transporter genes which have been associated with cardiac hypertrophy and failure in vivo. NRVM were maintained in serum-free culture medium under control conditions, or subjected to cyclic mechanical deformation (1.0 Hz, 20% maximal strain, 48 h). Under these conditions, cyclic stretch induced NRVM hypertrophy, as evidenced by significant increases in total protein/DNA ratio, myosin heavy chain (MHC) content, and atrial natriuretic factor (ANF) secretion. Cyclic stretch also induced the MHC isoenzyme "switch" which is characteristic of hemodynamic overload of the rat heart in vivo. Cyclic stretch significantly down-regulated SERCA2 and ryanodine receptor (RyR) mRNA and protein levels, while simultaneously increasing ANF mRNA. In contrast, Na+-Ca2+ exchanger and phospholamban mRNA levels were unaffected. Load-dependent SERCA2 and RyR down-regulation was independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels, as cyclic stretch down-regulated SERCA2 and RyR mRNA levels in both control and verapamil-treated NRVM. These results indicate that extrinsic mechanical load (in the absence of other exogenous stimuli) induces NRVM hypertrophy and causes down-regulation of Ca2+ transporter gene expression. This in vitro model system should prove useful to dissect the intracellular signaling pathways responsible for transducing this phenotype during cardiac hypertrophy and heart failure in vivo.     

A high resolving power ion selector for post-source decay measurements in a reflecting time-of-flight mass spectrometer

An electrostatic deflector has been designed and constructed that can be used in a reflecting time-of-flight mass spectrometer for either single-deflector or dual-deflector velocity selection in post-source decay measurements. The deflector consists of an interleaved set of parallel deflection electrodes as in a Loeb/Cravath/Bradbury device, but thin metal ribbon instead of wire is used for the deflection electrodes. The time for reversing the electric field, which depends on various factors such as the electronics for pulsing the voltage and the time constant of a particular electrode geometry, is about 19 ns for the deflectors used in this study. By properly timing the reversal of the electric field, the time-window for ion transmission can be made substantially less than the switching time of each individual deflector. In conjunction with matrix-assisted laser desorption/ionization, the single-deflector's resolving power and transmission are robust with respect to laser fluence, i.e. they remain high even when the fluence is raised well above threshold. By contrast the operational features of the dual-deflector gate offer more versatility in locating and sizing the selection window. Operating the ion selector in a single-deflector mode, we have achieved a resolving power of approximately 710 full width at half maximum (FWHM) for different isotopes of protonated, sodiated, and potassiated substance-P (m/z 1348.6, 1370.6 and 1386.6 respectively; 10.073 keV). Operating it in the dual-deflector mode under two different sets of conditions, we have succeeded in obtaining resolving powers of approximately 1100 (FWHM) for protonated substance-P (m/z 1348.6; 10.8 keV) and approximately 5200 (FWHM) for an isotopomer of PEG 6000 (approximately m/z 6000; 10.04 keV). This accomplishment implies that high-resolution ion selection can be coupled to post-source decay analyses.     

Concurrent use of cocaine and alcohol is more potent and potentially more toxic than use of either alone--a multiple-dose study

BACKGROUND: Simultaneous abuse of cocaine and alcohol is widespread and increasingly detected in patients seeking emergent care. This double-blind, randomized, within-subjects study used a paradigm more closely approximating practices of drug abusers to better understand the pathogenesis of cocaine-alcohol abuse. METHODS: Subjects meeting DSM-IV criteria for cocaine dependence and alcohol abuse participated in three drug administration sessions: four doses of intranasal cocaine (1 mg/kg every 30 min) with oral alcohol (1 g/kg) administered following the initial cocaine dose and a second dose (120 mg/kg) at 60 min calculated to maintain plasma alcohol concentration at approximately 100 mg/dL during cocaine administration; four doses of cocaine/placebo alcohol; four doses of cocaine placebo/alcohol. Pharmacokinetic, physiological, and behavioral effects were followed over 8 hours. RESULTS: Cocaine-alcohol produced greater euphoria and increased perception of well-being relative to cocaine. Heart rate significantly increased following cocaine-alcohol administration relative to either drug alone. Cocaine concentrations were greater following cocaine-alcohol administration. Cocaethylene had a longer halflife with increasing concentrations relative to cocaine at later time points. CONCLUSIONS: Enhanced psychological effects during cocaine-alcohol abuse may encourage ingestion of larger amounts of these substances over time placing users at heightened risk for greater toxicity than with either drug alone.     

Microbial populations of Botrytis cinerea-inoculated strawberry fruit exposed to four volatile compounds

OBJECTIVE: The authors tested the hypothesis that eye tracking disorder in schizophrenia is associated with neurological signs. METHOD: The subjects were 93 normal comparison subjects and 59 schizophrenic patients. They were evaluated with the Neurological Evaluation Scale, a standardized rating instrument that assesses sensory integration, motor coordination, sequencing of complex motor acts, and other neurological signs. Also, the schizophrenic patients' smooth-pursuit eye movements were tested in response to a 0.3-Hz sinusoidal target by means of infrared oculography. They were divided into those with (N=18) and without (N=41) eye tracking disorder by using a previously described method, which was based on mixture analysis of the distribution of position root mean square error. RESULTS: The patients with eye tracking disorder had significantly worse performance than the patients without eye tracking disorder with respect to sensory integration, and the effect size was moderate to large. In comparison with the normal subjects, both patient subgroups had significantly worse performance on all of the Neurological Evaluation Scale subscales. CONCLUSIONS: Although neurological signs are present generally in schizophrenia, poor sensory integration is particularly pronounced in patients with eye tracking disorder. A review of the literature shows that the two abnormalities have strikingly similar patterns of validators, including 1) familial aggregation, 2) premorbid presence, 3) syndromal specificity, 4) trait status, and 5) association with the deficit syndrome. Poor sensory integration and eye tracking disorder in schizophrenia may be various manifestations of a common, underlying pathophysiological process.     

Relationships among HIV risk beliefs, attitudes, and behaviors in sexually active, seronegative gay men

Safer-sex guidelines established during the early days of the HIV/AIDS epidemic have undergone very little revision despite some controversy. Although these guidelines have been widely disseminated in the gay community, many gay men continue to engage in behaviors that are believed to put them at high risk for transmission of HIV. This suggests either that they have not accepted safer-sex guidelines as accurate or that other factors override personal implementation of the guidelines. The study examined seronegative gay men's beliefs about the accuracy of safer sex guidelines and the men's participation in behaviors risky for transmitting HIV. The greatest disagreement between the men's beliefs and behaviors centered on the risk of oral intercourse; this suggests a need for clarification of the safer sex message about this behavior. The findings of this study support the need to reformulate safer-sex guidelines. When unprotected oral and anal sex are classified at the same level of risk, those who engage in unprotected oral sex may proceed to unprotected anal sex with less reservation.     

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sj?gren's syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sj?gren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sj?gren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sj?gren's syndrome.