Social phobia when using a second language.
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Humans↗
Biomedical subjects
A Schmidt
Publications and source records attributed to A Schmidt.
Serial in vivo passage of HIV-1 infection in Macaca nemestrina.
In an earlier study we found that pigtailed macaques (Macaca nemestrina) that were experimentally infected with human immunodeficiency virus type 1 (HIV-1) initially became viremic and seroconverted, but HIV-1 replication diminished markedly over time. In an attempt to develop a longer term pathogenic model, blood from HIV-1-infected macaques was serially transfused into three groups of naive macaques. Transfer was successful through two transfusions as shown by repeated virus isolations and confirmed by the development of cell-free plasma viremia and by seroconversion. Three to five weeks after transfusion, plasma levels of HIV-1 RNA from several macaques in the first two groups exceeded those of the initially inoculated macaques. However, animals in the third group had diminished RNA levels, were virus culture negative, and did not seroconvert. Sequence analyses of env-region clones from infected animals revealed only minimal changes over the course of the passages. These results confirm HIV-1 replication in M. nemestrina during the acute phase of infection. However, adaptation of HIV-1 to a macaque-pathogenic variant did not occur during serial passage, possibly because the animals were able to restrict HIV-1 replication below a level required for a pathogenic variant to emerge. Whether such containment is a function of the host's immune response or a virus cell incompatibility remains to be determined.
Adaptation, Physiological↗
DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.
The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.
Bacterial Proteins↗
Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.
Reports of significant reductions in plasma viral load by anti-HIV drugs have raised the possibility that antiviral therapy, if initiated sufficiently early, may result in sustained control of infection and prolonged clinical benefits. We evaluated the effects of intervention coincident with infection using an antiviral nucleoside, d4T, in Macaca nemestrina infected with a highly pathogenic isolate of HIV-2 (HIV-2[287]). Infection with this virus reproducibly results in high viremia and rapid CD4+ cell depletion, allowing a sensitive measurement of the treatment effect on viral load and clinical outcome. Compared to the control group, d4T-treated macaques showed significantly lower (2-3 log10) plasma- and cell-associated viral load. No CD4+ cell decline was observed in the treatment group while on therapy with d4T whereas CD4+ cells of control macaques declined from a preinfection mean of 32% of PBMCs to below 10%. Notably, when d4T treatment was withdrawn after 16 weeks, five of the six macaques continued to control viral load and have maintained normal CD4+ cell level for more than a year. These results demonstrate that early antiviral intervention, even of a limited duration, may constitute an important strategy against lentiviral-induced disease.
Animals↗
Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor.
To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.
Animals↗
Alendronate inhibition of protein-tyrosine-phosphatase-meg1.
Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is a potent bisphosphonate that inhibits osteoclastic bone resorption and has proven effective for the treatment of osteoporosis. Its molecular mechanism of action, however, has not been defined precisely. Here we report that alendronate is a potent inhibitor of the protein-tyrosine-phosphatase-meg1 (PTPmeg1). Two substrates were employed in this study: fluorescein diphosphate and the phosphotyrosyl peptide src-pY527. With either substrate, alendronate was a slow binding inhibitor of PTPmeg1. Among the other bisphosphonates studied, alendronate was more potent and selective for PTPmeg1. The hydrolysis of fluorescein diphosphate by PTP epsilon and PTPmeg1 was sensitive to alendronate, with IC50 values of less than 1 microM; PTPsigma, however, under the same conditions, was inhibited by only 50% with 141 microM alendronate. Similarly, with the src-pY527 substrate, alendronate inhibition was also PTP dependent. Alendronate inhibited PTPmeg1 with an IC50 value of 23 microM, PTPsigma with an IC50 value of 2 microM, and did not inhibit PTP epsilon at concentrations up to 1 mM. The alendronate inhibition of these three PTPs and two substrates is consistent with the formation of a ternary complex comprised of enzyme, substrate, and inhibitor. PTP inhibition by hisphosphonates or vanadate was diminished by the metal chelating agent EDTA, or by the reducing agent dithiothreitol, suggesting that a metal ion and the oxidation of a cysteine residue are required for full inhibition. These observations show substrate- and enzyme-specific PTP inhibition by alendronate and support the possibility that a certain PTP(s) may be the molecular target for alendronate action.
Alendronate↗
Sequence-specific antiproliferative effects of antisense and end-capping-modified antisense oligodeoxynucleotides targeted against the 5'-terminus of basic-fibroblast-growth-factor mRNA in coronary smooth muscle cells.
Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 microM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 microM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nonbase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF-specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post-angioplasty restenosis.
Animals↗
Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p.
BACKGROUND: The pathogenic fungus Candida albicans is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of mycelia displaying hyphae with branches and lateral buds. Hyphae have been observed to adhere to and invade host tissues more readily than the yeast form, suggesting that filamentous growth may contribute to the virulence of this major human pathogen. A molecular and genetic understanding of the potential role of morphological switching in the pathogenicity of C. albicans would be of significant benefit in view of the increasing incidence of candidiasis. RESULTS: The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of cells of the budding yeast Saccharomyces cerevisiae deleted for the STE20 gene and the CLA4 gene. CaCLA4 encodes a member of the Ste20p family of serine/threonine protein kinases and is characterized by a pleckstrin homology domain and a Cdc42p-binding domain in its amino-terminal non-catalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vitro, in both synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The gene deletions reduced colonization of the kidneys in infected mice and suppressed C. albicans virulence in the mouse model. CONCLUSIONS: Our results demonstrate that the function of the CaCla4p protein kinase is essential for virulence and morphological switching of C. albicans in a mouse model. Thus, hyphal formation of C. albicans mediated by CaCla4p may contribute to the pathogenicity of this dimorphic fungus, suggesting that regulators of morphological switching may be useful targets for antifungal drugs.
Amino Acid Sequence↗
Constitutive expression of a CD44 variant isoform on T cells facilitates regaining of immunocompetence in allogeneic bone marrow transplantation.
Constitutive expression of a rat CD44 variant isoform, rCD44v4-v7, on murine T cells accelerates immune responsiveness. Because prolonged immunodeficiency can be a major drawback in allogeneic bone marrow transplantation, we considered it of special interest to see whether repopulation of lethally irradiated syngeneic and allogeneic mice may be influenced by constitutive expression of the rCD44v4-v7 transgene. When lethally irradiated syngeneic and allogeneic mice were reconstituted with bone marrow cells (BMC) from rCD44v4-v7 transgenic (TG) or nontransgenic (NTG) mice, the former had a clear repopulation advantage: thymocytes expanded earlier after reconstitution and, as a consequence, higher numbers of lymphocytes were recovered from spleen and lymph nodes. Lymphocytes also displayed functional activity in advance to those from mice reconstituted with BMC from NTG mice. Most importantly, after the transfer of BMC from TG mice into an allogeneic host, the frequency of host-reactive T cells decreased rapidly. Apparently, this was due to accelerated induction of tolerance. Because these effects were counterregulated by an rCD44v6-specific antibody, it is likely that they could be attributed to the rCD44v4-v7 TG product. Thus, expression of a CD44 variant isoform at high levels facilitated reconstitution with allogeneic BMC by accelerated establishment of tolerance and the regaining of immunocompetence.
Animals↗
Synthesis and oral efficacy of a 4-(butylethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active corticotropin-releasing factor1 receptor antagonist.
The syntheses of a centrally active nonpeptide CRF1 receptor antagonist 2, butylethyl[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo [2,3-d]pyrimidin-4-yl]amine (CP-154,526), and its analogs 11-14 and [3H]-2 are reported. The in vitro CRF1 receptor binding affinity in the series 2, the pharmacokinetic properties of 2 in rats, and the anxiolytic-like effects of orally administered 2 are presented.
Animals↗
Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.
We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.
Animals↗
Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells.
Renal secretion of organic cations involves at least two distinct transporters, located in the basolateral and apical membranes of proximal tubule cells. Whereas the basolateral transporter has recently been cloned, sequence information about the apical type was not yet available. An organic cation transporter, OCT2p, was cloned from LLC-PK1 cells, a porcine cell line with properties of proximal tubular epithelial cells. OCT2p was heterologously expressed and characterized in human embryonic kidney 293 cells. OCT2p-mediated uptake of the prototypical organic cation [14C]tetraethylammonium ([14C]TEA) into 293 cells was saturable. There was a highly significant correlation between the Ki values for the inhibition of apical [14C]TEA uptake into LLC-PK1 cells and 293 cells transfected with OCT2p (r = 0.995; p < 0.001; n = 6). Although OCT2p is structurally related to OCT1r, the basolateral organic cation transporter from rat kidney, the transporters could be clearly discriminated pharmacologically with corticosterone, decynium22, and O-methylisoprenaline. The findings at hand suggest that OCT2 corresponds to the apical type of organic cation transporter. Reverse transcriptase-polymerase chain reaction indicates that mRNA of OCT1r is limited to non-neuronal tissue, whereas OCT2r, the OCT2p homologue from rat, was found in both the kidney and central nervous regions known to be rich in the monoamine transmitter dopamine.
Amino Acid Sequence↗
The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2.
The Saccharomyces cerevisiae phosphatidylinositol kinase homolog TOR2 is required for organization of the actin cytoskeleton. Overexpression of RHO1 or RHO2, encoding Rho-like GTPases, or ROM2, encoding a GDP/GTP exchange factor for RHO1 and RHO2, suppresses a tor2 mutation. Deletion of SAC7, a gene originally identified as a suppressor of an actin mutation, also suppresses a tor2 mutation. SAC7 is a novel GTPase-activating protein for RHO1. ROM2 exchange activity is reduced in a tor2 mutant, and overexpression of ROM2 lacking its PH domain can no longer suppress a tor2 mutation. Thus, TOR2 signals to the actin cytoskeleton through a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7. TOR2 activates this switch via ROM2, possibly via the ROM2 PH domain.
Cell Cycle Proteins↗
Species-specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes.
The secretory cells of the oviductal epithelium secrete a high-molecular-weight glycoprotein (OGP). OGPs from different mammalian species show similar immunological characteristics, their cDNAs show high homologies, and they associate with the zona pellucida of oviductal oocytes in vivo. The purpose of this study was to determine the effect of OGP obtained from different species on the binding of hamster sperm to hamster oocytes. Hamster oocytes were inseminated (30 min) in the presence or absence of homologous or heterologous OGPs, and sperm bound/oocyte were counted after removing loosely attached sperm. Ovarian oocytes had an average of 2.9 +/- 0.6 sperm bound/oocyte, whereas oviductal oocytes had 36.3 +/- 2.7. Hamster OGP (0.1 mg/ml) significantly increased sperm binding to ovarian oocytes twofold and had no effect on sperm bound/oviductal oocytes. Human OGP (0.5 mg/ml) significantly decreased sperm binding to ovarian oocytes (0.9 +/- 0.3 sperm bound/oocyte). This effect was dose dependent for oviductal oocytes and could be blocked by preincubating human OGP with a specific antibody to human OGP. The presence of baboon and cow OGP during the insemination of hamster oviductal oocytes also resulted in a significant decrease in sperm bound/oocyte, whereas the addition of hamster OGP to hamster oviductal oocytes had no effect. These results show that homologous OGP enhances sperm binding to the ZP, whereas heterologous OGP inhibits that effect. Thus, our results suggest that OGP plays a role in the species-specific characteristics of sperm/ZP interaction, and that one must use a homologous system (OGP and gametes from the same species) to study the biological effect of OGP.
Animals↗
Nitric oxide does not reverse pulmonary hypertension associated with end-stage liver disease: a preliminary report.
Pulmonary hypertension is a well known, though uncommon complication of end-stage liver disease (ESLD). Patients with severe pulmonary hypertension and ESLD undergoing orthotopic liver transplantation (OLT) may develop right ventricular failure and death. This study investigates the reversibility of pulmonary hypertension by the inhalation of nitric oxide in patients under evaluation for OLT. Ten patients with ESLD who were discovered to have moderate to severe pulmonary hypertension were administered nitric oxide via face mask in concentrations ranging from 0 to 80 ppm. Inhaled nitric oxide is a potent pulmonary vasodilator without apparent systemic effects. Nitric oxide had no demonstrable effect on mean pulmonary artery pressure (PAP) (37 vs. 37 mm Hg), transpulmonary gradient (TPG) (26 vs. 26 mm Hg), or pulmonary vascular resistance (PVR) (295 vs. 288 dynes x sec x cm (-5)). Two patients were discovered to have an elevated pulmonary artery occlusion pressure (PAOP) on baseline readings. The cause of pulmonary hypertension in these two patients was secondary to volume overload as a result of hepato-renal syndrome rather than primary pulmonary arteriolar pathology and was responsive to diuresis or dialysis but not to nitric oxide therapy. In conclusion nitric oxide does not reverse pulmonary hypertension associated with ESLD.
Administration, Inhalation↗
[Duodenum preserving resection of the head of the pancrease in therapy of pancreas divisum].
In 1-6% of the patients who are investigated by endoscopic retrograde cholangio-pancreatography a pancreas divisum can be found. In some patients pancreas divisum can lead to an acute relapsing and finally chronic pancreatitis (CP). Surgical intervention in these cases seems to offer a good chance of recovery. We report our experience with the duodenum-preserving resection of the head of the pancreas in 12 patients with pancreas divisum and CP. In all patients the preoperative evaluation showed clinical, functional or radiological signs of CP. The duodenum-preserving resection of the head of the pancreas was carried out in all patients without perioperative mortality. Ten postoperative versus eight preoperative patients showed a pathological exocrine function of the pancreas. Endocrine function, measured by the oral glucose tolerance test (OGTT), improved postoperatively in two patients. Eleven patients who were investigated after a mean follow-up time of 31 months (3-75 months) were completely pain free. No late mortality occurred. OGTT revealed a diabetic endocrine function in two patients. Disturbed exocrine pancreatic function had to be substituted in nine patients. One patient had to be reoperated by duct incision and renewal of the pancreatico-jejunostomy 10 months after the first operation. In conclusion, the duodenum-preserving resection of the head of the pancreas reduced pain in all patients with pancreas divisum and CP and may lead to an improvement of endocrine pancreatic function. Other, nonresecting procedures or endoscopic interventional therapy should be avoided in these patients.
Adult↗
Evaluation of risk factors for the development of nephropathy in patients with IDDM: insertion/deletion angiotensin converting enzyme gene polymorphism, hypertension and metabolic control.
Diabetic nephropathy represents a major complication in patients with insulin-dependent diabetes mellitus (IDDM). Intervention trials using angiotensin-converting enzyme (ACE) inhibitors have pointed towards the important pathogenetic role of the renin-angiotensin system. Recently an insertion/ deletion (I/D) polymorphism for the gene encoding the ACE has been described, the deletion type being associated with higher plasma ACE levels. As the intrarenal renin-angiotensin system might also be activated in this setting, we determined the ACE genotype together with other risk factors for the development of diabetic nephropathy in 122 patients with IDDM from a single centre with (n = 63) and without (n = 59) nephropathy. Long-term glycaemic control was evaluated using mean HbA1c values from the last 10 years. The two patient group were comparable with regard to duration of diabetes and glycaemic control as assessed by current HbA1c values. However, mean long-term HbA1c values were significantly higher in patients with diabetic nephropathy as was systemic blood pressure. The DD genotype was more prevalent in patients with renal disease. In the subgroup of patients who had had diabetes for more than 20 years (n = 90), the DD genotype was even more frequent in patients with nephropathy, and blood pressure and long-term HbA1c values were also higher in patients with renal disease. Logistic regression analysis revealed long-term glycaemic control, blood pressure and the ACE genotype to be independent risk factors for the prevalence of diabetic nephropathy.
Blood Glucose↗
Effect of 3 hours' passive smoke exposure in the evening on airway tone and responsiveness until next morning.
To study the effect of environmental tobacco smoke (ETS) exposure in the evening on nocturnal changes in airway tone and responsiveness, 17 subjects with mild asthma (mean +/- SD age, 26 +/- 5 years, FEV1% pred., 89 +/- 14%) were exposed to either ambient air (sham) or ETS (20 ppm CO) for 3 h (7:00 to 10:00 p.m.). Seven subjects had a history of ETS-induced respiratory symptoms. Spirometry was performed 2 h before exposure (5:00 p.m.), every 30 min during exposure, and at 11:00 p.m., 3:00 a.m., and 7:00 a.m. The provocative concentrations of methacholine necessary to decrease FEV1 by 20%, PC20FEV1, were assessed at 5:00 p.m., 11:00 p.m., 3:00 a.m., and 7:00 a.m. Compared with pre-exposure measurements, mean FEV1 values during and after ETS exposure were significantly lower than with sham exposure = 0.013 and 0.026). This effect, however, was due to a significant response in single individuals. The higher bronchial responsiveness after ETS than after sham exceeded one doubling concentration in 4, 5, and 4 patients at 11:00 p.m., 3:00 a.m., and 7:00 a.m., respectively. The opposite effect was observed in 2, 2, and 2 patients, respectively. There was no statistically significant mean effect of ETS on airway responsiveness during night; however, there was significant heterogeneity in individual responses (P = 0.0002). Patients with and without a history of ETS-induced symptoms did not show different responses to experimental ETS exposure. In conclusion, our data suggest that in single adult subjects with mild asthma, acute exposure to ETS in the evening can produce a deterioration of airway tone and responsiveness during the night, with wide interindividual variability in the response.
Adult↗