The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry.

The complexity of signal transduction resulting from the contact of human immunodeficiency virus type 1 (HIV-1)-infected cells and mucosal cells has hampered our comprehension of HIV-1 mucosal entry. Such process is driven efficiently only by viral synapse contacts, whereas cell-free HIV-1 remains poorly infectious. Using CD4+ T-cells expressing only HIV-1 envelope inoculated on human adult foreskin tissues, we designed methodologies to identify the signals transduced in foreskin keratinocytes following HIV-1-envelope-dependent viral synapse formation. We find that the viral synapse activates the MyD88-independent TLR-4-nuclear factor (NfκB) signaling pathway in keratinocytes and the subsequent secretion of cytokines including thymic stromal lymphopoietin (TSLP), a cytokine linking innate and T-helper type 2-adaptive immune responses. Moreover, the viral synapse upregulates the non-coding microRNA miR-375, known to control TSLP, and transfection of keratinocytes with anti-miR-375 blocks significantly TSLP secretion. Thus, the secretion of TSLP by keratinocytes is induced by the viral synapse in a miR-375 controlled manner. At the tissue level, these signals translate into the epidermal redistribution of Langerhans cells and formation of conjugates with T-cells, recapitulating the initial events observed in human foreskin infection by HIV-1. These results open new possibilities for designing strategies to block mucosal HIV-1 transmission, the major pathway by which HIV-1 spreads worldwide.

Body growth, hematological profile, and clinical biochemistry of heifer calves sired by a bull or its clone.

The aim of this paper was to compare body growth, hematological profile development, and clinical biochemistry in the female progeny of a sire with the female progeny of its clone. Sixteen Friesian female calves, 9 daughters from a tested bull (BULL) and 7 from its somatic cell nuclear transfer clone (CLONE) were monitored from birth to 60 wk of life. Body weight (BW), wither height (WH), hip height (HH), body length (BL), and hearth girth (HG) were measured at birth and 4, 8, 12, 16, 20, 24, 36, and 50 wk. Blood samples were taken from jugular vein at 12 to 48 h from birth and 1, 2, 3, 4, 8, 12, 16, 20, 24, and 36 wks of age, to be analyzed for hematological, serum protein, and metabolic profiles. At the same time, rectal temperature (RT) was recorded. Age at puberty was assessed on surviving heifers by measuring weekly plasma progesterone levels. Data were evaluated using a mixed model, taking into account the repeated measures in time on the calf. For each variable, different covariance structures were tested, choosing the best according to the Akaike's Information Criteria. Significant was set at P < 0.05, and a trend was considered for P < 0.10. At 24 wk of age, WH was lower in CLONE daughters than BULL daughters. Around 20 wk of age, there was a trend for lower BW in CLONE daughters than BULL daughters, confirmed from differences in HG. There was no difference in RT due to sire effect. Blood glucose concentration decreased in both groups during the first 4 wk of life; at birth, only a trend for higher blood glucose in CLONE daughters was recorded, whereas an opposite trend was observed for plasma creatinine. Total leukocyte count did not differ between progenies. Circulating lymphocytes tended to be lower in CLONE than BULL daughters. The neutrophil: lymphocyte ratio tended to be higher in CLONE than BULL calves. No difference was demonstrated for erythrocyte features, whereas mean platelet volume tended to be lower in CLONE than BULL progeny. From these results, there were no differences between progenies from BULL and its clone that suggest welfare problems in the first 6 mo of life.

Formulation development and human in vitro-in vivo correlation for a novel, monolithic controlled-release matrix system of high load and highly water-soluble drug niacin.

Novel, controlled-release formulations for high drug load, highly water soluble compound niacin based on polyethylene oxide (PEO) and hydroxypropylmethyl cellulose (HPMC) matrices were developed and investigated. The effect of sodium bicarbonate as a modulator of swelling, erosion, and drug release and its impact on changes in the kinetics of axial swelling and gel strength were evaluated by textural analysis during dissolution study. The drug release rate from PEO-based matrices was faster and correlated with lower gel strength, greater water uptake, and greater matrix erosion. Slower release rate and greater release duration correlated significantly with greater matrix swelling with negligible matrix erosion for the HPMC-based matrix system. Inclusion of sodium bicarbonate in the polymeric matrix salted out the macromolecules and increased gel strength and gel viscosity, especially in the vicinity of the swelling fronts. An in vivo study in human subjects after administration of the formulations and a commercial product exhibited similar plasma concentrations. For the formulation of interest, the mean drug fraction absorbed by the body was calculated by the Wagner-Nelson technique, and a level A "in vitro-in vivo correlation" was observed between the percent released in vitro and percent absorbed in vivo. The developed formulations appear to be robust and easy to manufacture with maximum flexibility with respect to drug dose, polymeric carriers, duration, and kinetics of drug release.

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes.

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.

Degradation of historical paper: nondestructive analysis by the NMR-MOUSE.

The NMR-MOUSE is a mobile sensor for single-sided NMR inspection of organic materials which takes advantage of the principles of magnetic resonance and inside-out-NMR. Historical books dating from the 17th century were measured at different points by positioning the NMR-MOUSE on the paper. Different degrees of paper degradation can be discriminated from the regularized inverse Laplace transform of the envelope of the acquired echo signals. For the first time the degradation of historical paper was characterized entirely nondestructively by NMR. As a contribution to current preservation efforts, NMR shows great promise for future use in damage assessment of historical documents.

Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems.

Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30-31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme.

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.

Development of molecular markers linked to a gene controlling fruit acidity in citrus.

Fruit juice pH, titratable acidity, or citric acid content was measured in 6 populations derived from an acidless pummelo (pummelo 2240) (Citrus maxima (Burm.) Merrill). The acidless trait in pummelo 2240 is controlled by a single recessive gene called acitric. Using bulked segregant analysis, three RAPD markers were identified as linked to acitric. RAPD marker OpZ20410, which mapped 1.2 cM from acitric, was cloned and sequenced, and a sequence characterized amplified region (SCAR) marker (SCZ20) was developed. The SCZ20-410 marker allele that is linked to the acitric allele occurs only in pummelo 2240 and other pummelos, and therefore, this SCAR marker should be useful as a dominant or codominant marker for introgressing acitric into mandarins and other citrus species. Using the cloned OpZ20410 band as a hybridization probe revealed a codominant RFLP marker called RFZ20 that mapped 1.2 cM from acitric. Progeny homozygous (acac) for the acitric allele had citric acid content below 10 μM, the minimum level detectable by high pressure liquid chromatography. The citric acid content of fruit juice from progeny predicted to be heterozygous (Acac) for acitric by the above markers was about 30% lower than that of juice from individuals predicted to be homozygous (AcAc) for the normal acid allele. Markers OpZ20410, SCZ20, and RFZ20 were highly polymorphic among 59 citrus accessions, and using one or more of these markers would allow citrus breeders to select seedling progeny heterozygous for acitric in nearly all crosses between pummelo 2240 or its offspring and other citrus genotypes.

Preconcentration of heavy metals ions from aqueous solutions by means of cellulose phosphate: an application in water analysis.

Cellulose phosphate (CELLPHOS) was studied as a collector for analytical preconcentration of traces of Cd(II), Cr(III), Cu(II) and Ni(II) from aqueous sample solution. It has been proved that using chromatographic columns packed with CELLPHOS for preconcentration and 1.0 mol 1(-1) HCl for elution the adsorbed analytes are quantitatively enriched. An enrichment factor of 20 (100 ml sample, 5 ml concentrate) was achieved by this separation procedure, which was applied to a series of water analyses (river, sea, bog water).

Association of the cytoplasmic domain of intercellular-adhesion molecule-1 with glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin.

To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule-1 (ICAM-1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27-amino-acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM-1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM-1-interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein beta-tubulin and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraP-DH). Experiments carried out with purified GraP-DH or CNBr fragments of GraP-DH indicated that binding to the ICAM-1 matrix was mediated by the C-terminal domain of GraP-DH, containing the binding site of the cofactor NAD+, and that NAD+ could compete with this binding. Using a series of ICAM-1 C-terminal truncated peptides, we could demonstrate that (a) the nitric-oxide-induced covalent linkage of NAD+ to GraP-DH was impaired by these peptides, (b) the glycolytic activity of GraP-DH was drastically inhibited by a truncated peptide containing the 15 C-terminal residues, (c) nitric oxide appeared to prevent this inhibition. Together, our results demonstrate that GraP-DH specifically associates with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.

Predictive quantitative structure-activity relationships (QSAR) analysis of beta 3-adrenergic ligands.

A novel quantitative structure-activity relationships strategy was used to analyze seventeen beta-adrenergic ligands for which we had previously evaluated pharmacological properties in Chinese hamster ovary cells transfected with the human beta 1-, beta 2- or beta 3-adrenergic gene (Blin et al., 1993, Mol. Pharmacol., 44: 1094-1104). These ligands were classified into pharmacological activity categories in order to determine the extent to which molecular structural features may be involved in the selectivity of the interaction with the beta 3-AR, or to define molecular features and properties characteristic of a beta 3-AR high affinity ligand or of a potent beta 3-adrenergic agonist. Topological and physico-chemical molecular descriptors were obtained using a novel software combining calculations with multivariate statistical methods, such as principal component analysis and discriminant analysis. This study showed that beta 1/beta 2-antagonists beta 3-agonists could be differentiate from beta 1/beta 2/beta 3-agonists on the basis of their topological molecular descriptors weighted by partial atomic charge and lipophilicity logP values. Bulky lipophilic groups at the end of the alkylamine chain and an ethoxy function, extending the flexible portion of the molecule and modifying the electron density distribution, were requirements for selective agonism at the beta 3-site. Charge and logP weighted 2D-autocorrelation vectors were properties able to discriminate between classes of agonists in terms of their affinity, potency or intrinsic activity, thus emphasizing the part these molecular descriptors play in determining beta 3-adrenergic ligands. These results, in association with the powerful activity-prediction model evaluated in the test, provide a framework to rationalize the synthesis of new beta 3-AR specific compounds.

Cultured astrocytes release a factor that decreases endothelin-1 secretion by brain microvessel endothelial cells.

Endothelin-1 (ET-1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood-brain barrier, have been shown to secrete ET-1, whereas specific ET-1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET-1 and its precursor big ET-1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET-1 production was purified by gel-exclusion and ion-exchange chromatography as a 280-Da iron-containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron-nitrogen oxide complex, may be involved in a regulatory loop of ET-1 production at the level of the blood-brain barrier.

Nitric oxide and endothelin secretion by brain microvessel endothelial cells: regulation by cyclic nucleotides.

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFN gamma and TNF alpha, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1.

The assessment and management of panic disorder.

Panic disorder affects approximately 1.5 percent of the general population in the United States, and individuals with this disorder are seen frequently in primary care settings. Recognition of panic disorder is complicated by the fact that patients tend to focus exclusively on the physical symptoms of the illness. Left untreated, patients with panic disorder suffer considerable impairment in functioning and are at increased risk for substance abuse, depression and suicide. This article provides clinicians with the information necessary to recognize and manage this illness. A detailed description of the disorder, including onset, course, complications, differential diagnosis and management, is presented in this article.

Screening for cancer and coronary risk factors through a nurse practitioner-staffed preventive health clinic.

The Preventive Health Examination (PHE) program was designed to screen for cancer of the lung, colon, skin, and prostate (or breast) and to detect the risk factors for coronary artery disease, i.e., arterial hypertension, hypercholesterolemia, cigarette smoking, and obesity. To investigate the cost-effectiveness of PHE performed by nurse practitioners, the first 176 subjects enrolled in the PHE program at a northeastern, suburban VA Medical Center were evaluated prospectively. The subjects were recruited through random mailing. The direct cost of PHEs was $80 per patient. PHEs were performed on 171 men and 5 women, mean age 57.2 years, divided into groups according to the date of evaluation. Nine percent of patients had findings highly suspicious of cancer (lung nodules in 7, skin lesions in 9). As a consequence, one patient underwent lobectomy for squamous carcinoma of the lung and another underwent prostatectomy for adenocarcinoma of the prostate. Fifty-five percent of patients had high cholesterol levels, 53% were obese, 30% were heavy cigarette smokers, and 19% were hypertensive. Nineteen percent of the patients had three or more coronary artery disease risk factors. We conclude that low cost PHEs performed by nurse practitioners have a high yield in detecting risk factors for coronary artery disease, and for detecting potentially treatable malignancies.

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM-CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

Endogenous factors possessing lymphoid chalone properties, purified by chromatography.

Lymphoid chalone extracts, obtained from bovine spleen were purified on chromatographic columns: in the first step of the procedure, exclusion chromatography on Sephadex G75, and ion exchange chromatography on DEAE Sephadex were applied. A purified, active material was thus obtained. Upon thin layer chromatography, this fraction was shown to contain 3-4 UV absorbing components and 2 ninhydrin positive components. Further purification on Biogel P2, on Sephadex G10 and preparative thin layer chromatography, showed that the biological activity was located in small molecular weight components which belong to the polypeptide series. The ultraviolet absorbing components were identified as known nucleosides. The purified material shows an inhibitory effect on the uptake of 3H-thymidine by lymphocytes, as well as on mitogen induced lymphocyte transformation and haemolysin plaque forming cells. Furthermore, this inhibitory effect is specific for lymphoid cells.