Citations

Transforming growth factor-β (TGF-β) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K+depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the μ2 subunit, AP2/TGF-β receptor binding was mediated by a direct interaction between the β2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the μ2, α, or ς2 subunits of AP2 or to μ1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of β2-adaptin to directly couple TGF-β receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with β2-adaptin.

Objective— C-reactive protein (CRP) is a sensitive marker of inflammation and a prognostic marker in cardiovascular disease. Evidence suggests direct biological activities of CRP within the vascular wall. The study was designed to examine the vasoreactive effects of CRP. Methods and Results— Human internal mammary artery rings were obtained during cardiovascular bypass surgery and suspended in an organ bath chamber. The rings were precontracted with endothelin-1, and response to cumulative concentrations of CRP was obtained. Experiments were repeated after initial incubation with 20, 40, and 60 mmol/L KCl, the potassium channel blockers BaCl, tetraethylammonium chloride, and glibenclamide, and the NO synthase inhibitor N -monomethyl- l -arginine and also after removal of the endothelium. CRP caused dose-dependent relaxation of human internal mammary artery rings, which was not affected by preincubation with N -monomethyl- l -arginine or removal of the endothelium. Maximum relaxation response to CRP (79.5±10%) was attenuated by KCl (2.5±11.5%, P <0.001), BaCl (24.5±7.5%, P <0.001), and tetraethylammonium chloride (34.9±8.25%, P <0.01) but not by glibenclamide. Conclusions– The present study demonstrates that CRP exerts an endothelium-independent vasorelaxing effect via potassium channels. Thus, the study suggests a role of CRP in the regulation of vascular tone.

ABSTRACT Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis -acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans . When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5′ leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.

ABSTRACT Although Helicobacter pylori is a cosmopolitan colonizer of the human stomach, the responses among persons in remote populations from whom H. pylori was cultured have not been studied. We report on studies of 189 persons in the Ladakh region of India in whom serum immunoglobulin G responses to H. pylori whole-cell and CagA antigens were measured. H. pylori was isolated from 68 of these patients. An H. pylori whole-cell antigen derived from Ladakhi strains outperformed a similar antigen from U.S. strains, as determined by antigen-specific enzyme-linked immunosorbent assays. In total, 95% of the population was seropositive, including individuals responding only to the CagA antigen. Correlation with culture results showed that these were true positives and, therefore, that the H. pylori whole-cell serology was falsely negative in some cases. In addition to establishing a collection of H. pylori isolates from a remote area in the world, we show that use of H. pylori whole-cell and CagA serology together increases the sensitivity for the detection of colonization.

Background and Purpose— The natural history of perihematomal edema in human hyperacute spontaneous intracerebral hemorrhage (ICH) has not been well described. Methods— This study was a secondary analysis of a previously reported prospective, population-based study of hematoma growth in 142 patients with spontaneous ICH. Patients were first imaged within 3 hours of onset, then 1 and 20 hours later. We excluded patients with anticoagulant use (n=7), underlying aneurysm/vascular malformation (n=9), trauma (n=1), incomplete data (n=20), infratentorial ICH (n=17), and no consent (n=2), leaving an overall study population of 86 patients. From this overall group we further excluded patients with intraventricular extension (n=38), subsequent surgery (n=5), or death (n=2) before 20-hour postbaseline CT. This second, “restricted” analysis group of 41 patients was relatively devoid of clinical or radiological variables likely to confound edema measurement. Absolute and relative edema volumes (edema volume divided by hematoma volume) were descriptively summarized. Correlations between baseline edema volumes and relevant clinical and radiological variables were then performed. Results— Overall, median absolute edema volume increased from 6.93 to 14.4 cm 3 during the first 24 hours after ICH, and median relative edema volume increased from 0.47 to 0.81. In the restricted group, median absolute edema volume was 7.4 cm 3 at baseline and 11.0 cm 3 at 24 hours after ICH, and median relative edema volume increased from 0.55 to 0.81. Baseline relative edema volume was significantly negatively correlated with subsequent change in relative edema volume from baseline to 20-hour CT ( r =0.57, P =0.0002) but was not significantly correlated with other clinical and radiological variables, including hematoma volume or change in hematoma volume. Conclusions— Perihematomal edema volume increases by approximately 75% during the first 24 hours after hyperacute spontaneous ICH. Patients with the least amounts of baseline relative edema volume were most likely to develop significant additional amounts of edema during the first 24 hours after spontaneous ICH.

Background and Purpose— Little is known about the relationship between perihematomal edema in spontaneous intracerebral hemorrhage (ICH) and outcome. The purpose of this study was to determine whether absolute or relative edema volume (edema volume divided by hematoma volume) predicts mortality or functional outcome in patients with hyperacute spontaneous ICH. We hypothesized that increasing baseline relative edema volume is associated with greater probability of poor functional outcome. Methods— This was a secondary analysis of a prospective, population-based study of hematoma growth in 142 patients with spontaneous ICH. Patients were imaged within 3 hours of onset, then 1 and 20 hours later. Our primary analysis excluded patients with anticoagulant use (n=7), underlying aneurysm/vascular malformation (n=9), trauma (n=1), incomplete data (n=20), infratentorial ICH (n=17), intraventricular extension (n=38), and no consent (n=2). We analyzed whether associations existed between baseline edema volumes or other clinical/radiological variables and either 12-week modified Rankin Scale score >2 or 30-day mortality. Secondary analyses used 20-hour CT scan data, all patients with supratentorial ICH, and 12-week Barthel Index score <85. Results— By multivariable logistic regression analysis, baseline relative edema was the strongest independent predictor of functional outcome and was associated with lesser odds of poor 3-month functional outcome (odds ratio, 0.09 per 1.0-unit [100%] increase; 95% CI, 0.01 to 0.64; P =0.016) and 12-week Barthel Index score <85 (odds ratio, 0.12; 95% CI, 0.02 to 0.91; P =0.039) but did not predict mortality. Secondary analyses confirmed this result. Absolute edema volume predicted neither mortality nor functional outcome. Conclusions— Relative edema is strongly predictive of functional outcome in patients with hyperacute supratentorial spontaneous ICH without intraventricular extension.

PURPOSE: Multiple myeloma is a malignancy of plasma cells and is characterized by increased marrow angiogenesis. Thalidomide, an agent with antiangiogenic properties, is effective in relapsed myeloma. We report the results of a study combining thalidomide and dexamethasone as initial therapy for myeloma. PATIENTS AND METHODS: Fifty patients with newly diagnosed myeloma were studied. Thalidomide was given at a dose of 200 mg/d orally. Dexamethasone was given at a dose of 40 mg/d orally on days 1 to 4, 9 to 12, and 17 to 20 (odd cycles) and 40 mg/d on days 1 to 4 (even cycles), repeated monthly. RESULTS: Of all 50 patients, a confirmed response was seen in 32 patients yielding a response rate of 64% (95% confidence interval, 49% to 77%). Thirty-one patients (62%) proceeded to stem-cell collection after four cycles of therapy including 26 who underwent stem-cell transplantation and five who chose stem-cell cryopreservation. Major grade 3 or 4 toxicities were observed in 16 patients (32%), and the most frequent were deep vein thrombosis (six patients), constipation (four patients), rash (three patients), and dyspnea (two patients). Three deaths occurred during active therapy because of a pancreatitis, pulmonary embolism, and infection. CONCLUSION: We conclude that the combination of thalidomide plus dexamethasone is a feasible and active regimen in the treatment of multiple myeloma. It merits further study as an oral alternative to infusional chemotherapy with vincristine, doxorubicin, and dexamethasone and other intravenous regimens currently used as pretransplantation induction therapy for myeloma.

ABSTRACT The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10 2 to 10 4 spores/ml of feces, a value which represented a significant improvement over that achieved by staining (≥1.0 × 10 6 spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species ( E . intestinalis , E . cuniculi , and E . hellem ). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

Background— Several scores, most of which were derived from clinical trials, have been proposed for stratifying risk after myocardial infarctions (MIs). Little is known about their generalizability to the community, their respective advantages, and whether the ejection fraction (EF) adds prognostic information to the scores. The purpose of this study is to evaluate the Thrombolysis in Myocardial Infarction (TIMI) and Predicting Risk of Death in Cardiac Disease Tool (PREDICT) scores in a geographically defined MI cohort and determine the incremental value of EF for risk stratification. Methods and Results— MIs occurring in Olmsted County were validated with the use of standardized criteria and stratified with the ECG into ST-segment elevation (STEMI) and non–ST-segment elevation (NSTEMI) MI. Logistic regression examined the discriminant accuracy of the TIMI and PREDICT scores to predict death and recurrent MI and assessed the incremental value of the EF. After 6.3±4.7 years, survival was similar for the 562 STEMIs and 717 NSTEMIs. The discriminant accuracy of the TIMI score was good in STEMI but only fair in NSTEMI. Across time and end points, irrespective of reperfusion therapy, the discriminant accuracy of the PREDICT score was consistently superior to that of the TIMI scores, largely because PREDICT includes comorbidity; EF provided incremental information over that provided by the scores and comorbidity. Conclusion— In the community, comorbidity and EF convey important prognostic information and should be included in approaches for stratifying risk after MI.