Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells

Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4(+) T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4(+) T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4(+) T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4(+) T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4(+) T-cell lines or primary CD4(+) T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4(+) T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4(+) T cells.     

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2

Background

Heterodimeric phospholipase A₂ from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity.

Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii

The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1' such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k(cat)/K(m)=2 x 10(5) m(-1) s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K(i)=1.5 nM), as well as a portion of the propeptide sequence, PT1 (K(i)=32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.     

Can micro-imaging based analysis methods quantify structural integrity of rat vertebrae with and without metastatic involvement?

This study compares the ability of μCT image-based registration, 2D structural rigidity analyses and multimodal continuum-level finite element (FE) modeling in evaluating the mechanical stability of healthy, osteolytic, and mixed osteolytic/osteoblastic metastatically involved rat vertebrae. μMR and μCT images (loaded and unloaded) were acquired of lumbar spinal motion segments from 15rnu/rnu rats (five per group). Strains were calculated based on image registration of the loaded and unloaded μCT images and via analysis of FE models created from the μCT and μMR data. Predicted yield load was also calculated through 2D structural rigidity analysis of the axial unloaded μCT slices. Measures from the three techniques were compared to experimental yield loads. The ability of these methods to predict experimental yield loads were evaluated and image registration and FE calculated strains were directly compared. Quantitatively for all samples, only limited weak correlations were found between the image-based measures and experimental yield load. In comparison to the experimental yield load, we observed a trend toward a weak negative correlation with median strain calculated using the image-based strain measurement algorithm (r=-0.405, p=0.067), weak significant correlations (p<0.05) with FE based median and 10th percentile strain values (r=-0.454, -0.637, respectively), and a trend toward a weak significant correlation with FE based mean strain (r=-0.366, p=0.09). Individual group analyses, however, yielded more and stronger correlations with experimental results. Considering the image-based strain measurement algorithm we observed moderate significant correlations with experimental yield load (p<0.05) in the osteolytic group for mean and median strain values (r=-0.840, -0.832, respectively), and in the healthy group for median strain values (r=-0.809). Considering the rigidity-based predicted yield load, we observed a strong significant correlation with the experimental yield load in the mixed osteolytic/osteoblastic group (r=0.946) and trend toward a moderate correlation with the experimental yield load in the osteolytic group (r=0.788). Qualitatively, strain patterns in the vertebral bodies generated using image registration and FEA were well matched, yet quantitatively a significant correlation was found only between mean strains in the healthy group (r=0.934). Large structural differences in metastatic vertebrae and the complexity of motion segment loading may have led to varied modes of failure. Improvements in load characterization, material properties assignments and resolution are necessary to yield a more generalized ability for image-based registration, structural rigidity and FE methods to accurately represent stability in healthy and pathologic scenarios.     

L-NAME does not affect exercise-induced pulmonary hypertension in Thoroughbred horses

The present study was carried out to examine theeffects of nitric oxide synthase inhibition withN-nitro-L-arginine methyl ester(L-NAME) on the right atrial as well as on the pulmonary arterial, capillary, and venous blood pressures of horses during rest and exercise performed at maximal heartrate (HR_max). Experiments werecarried out on seven healthy, sound, exercise-trained Thoroughbredhorses. Using catheter-tip manometers, with signals referenced at thepoint of the shoulder, we determined phasic and mean right atrial andpulmonary vascular pressures in two sets of experiments [control(no medications) and L-NAME (20 mg/kg iv given 10 min before exercise studies)]. The studies werecarried out in random order 7 days apart. Measurements were made atrest and during treadmill exercise performed on a 5% uphill grade at6, 8, and 14.2 m/s. Exercise on a 5% uphill grade at 14.2 m/s elicitedHR_max and could not be sustainedfor >90 s. In quietly standing horses,L-NAME administration caused asignificant rise in right atrial, as well as pulmonary arterial, capillary, and venous pressures. This indicates that nitric oxide synthase inhibition modifies the basal pulmonary vasomotor tone. Inboth treatments, exercise caused progressive significant increments inright atrial and pulmonary vascular pressures, but the values recordedin the L-NAME study were notdifferent from those in the control study. The extent ofexercise-induced tachycardia was significantly decreased in theL-NAME study at 6 and 8 m/s butnot at 14.2 m/s. Thus, L-NAMEadministration may not modify the equine pulmonary vascular tone duringexercise at HR_max. However, asindicated by a significant reduction in heart rate,L-NAME seems to modify thesympathoneurohumoral response to submaximal exercise.

     

Long-term serial cultivation of arterial and capillary endothelium from adult bovine brain

Summary Cerebrovascular endothelial cells from adult bovine brain were carried successfully in long-term, serial culture. Endothelial cells were obtained from the middle and anterior cerebral arteries and from capillaries isolated from grey matter of the cerebral cortex or caudate nucleus. Capillary cells were found to grow best in RPMI 1640 with 20% fetal bovine serum. They did not require tumor-conditioned medium or matrix-coated surfaces, although fibronectin was used to enhance the initial plating efficiency of the primary cultures. The same conditions were used to support satisfactory growth of arterial endothelial cells; however they did not grow as rapidly as the capillary cells. Retention of endothelial-specific characteristics were shown for capillary-derived cells carried up to Passage 28, arterial-derived cells up to Passage 11, and after frozen storage of both types of cultured cells. Cultures of both arterial and capillary cells stained positively for Factor VIII antigen, exhibited a nonthrombogenic surface, and produced prostacyclin in response to arachidonic acid. Arterial endothelial cells produced more prostacyclin than capillary endothelium. The capillary cells had a unique tendency to assume a ringlike morphology after subculture and sometimes formed capillarylike networks of cell cords in dense cultures. When cultured in a three-dimensional plasma clot, capillary and arterial endothelial cells, but none of the other cell types studied, organized into tubelike structures reminiscent of capillary formation in vivo. The availability of long-term cultures of cerebrovascular endothelial cells provides an opportunity to compare properties of arterial and capillary endothelium from the same tissue and to investigate such processes as angiogenesis and blood-brain barrier induction. This work was supported in part by Public Health Service grant NS-18586 from the National Institute of Neurological and Communicative Disorders and Stroke and by a grant from The Council for Tobacco Research—U.S.A., Inc. C. E. was supported by the Universidad Autonoma of Madrid and the Ministerio de Universidades e Investigacion of Spain.     

The carbon economy of drought: comparing respiration responses of roots,mycorrhizal fungi,and free-living microbes to an extreme dry-rewet cycle

Plant and Soil - The effects of drying and wetting on soil carbon processes are regulated by the responses of plants, plant-associated microbes, and free-living microbes. Whether these groups...