The binding characteristics of two monoclonal antibodies (mAb) to phosphatidylinositol-4-phosphate (PIP) were examined: a murine IgM mAb to PIP; and a human IgG mAb (4E10) that binds both to HIV-1 ...envelope protein and also to neutral and anionic phospholipids, including PIP. Binding of each mAb to pure PIP was inhibited by Ca
2+ as determined by ELISA. When studied by surface plasmon resonance, liposomes containing PIP could be stripped (i.e., removed) by either Ca
2+ or phosphorylated haptens after binding of the liposomes to the murine anti-PIP antibody attached to a BIAcore chip. In contrast, the binding of liposomal PIP to 4E10 was irreversible and could not be stripped. We therefore conclude that Ca
2+ and phosphate can modulate the initial binding of both types of antibodies to PIP. However, 4E10 binds to liposomal PIP in a two-stage process involving first Ca
2+-modulated binding to the PIP polar headgroup, followed by irreversible binding to liposomal hydrophobic groups.
The discoid form of blood platelets is important to their function in hemostasis. Recent studies have suggested that the spectrin-rich surface membrane cytoskeleton and the cytoplasmic, actin-rich ...cytoskeleton are responsible for discoid shape, shape change, and recovery after activation or chilling. Earlier studies had suggested that circumferential coils of microtubules supported the disc shape of resting platelets and that their repositioning or reassembly restored disc shape after exposure to low temperature. The present study has used the chilling-rewarming model, together with microtubule stabilizing (taxol) and disassembling (vincristine) agents to retest the relative importance of the surface membrane cytoskeleton and circumferential microtubules in platelet discoid shape and its restoration. Washed platelet samples were rested at 37 degrees C and chilled to 4 degrees C; chilled and rewarmed to 37 degrees C for 60 minutes; or chilled, rewarmed, and exposed to the same cycle in the presence or absence of vincristine or taxol and fixed for study by disseminated interference phase contrast microscopy and electron microscopy. Rhodamine-phalloidin and flow cytometry were used to measure changes in actin filament assembly. Chilling caused loss of disc shape, pseudopod extension, disassembly of microtubule coils, and assembly of new actin filaments. Rewarming resulted in restoration of disc shape, pseudopod retraction, disassembly of new actin filaments, and reassembly of circumferential microtubule coils. Vincristine converted discoid platelets to rounded cells that extended pseudopods when chilled and retracted them when rewarmed, leaving spheres that could undergo the same sequence of changes when chilled and rewarmed again. Taxol prevented cold-induced disassembly of microtubules and limited pseudopod formation. Rewarming caused retraction of pseudopods on taxol-treated, discoid cells. Cytochalasin B, an agent that blocks new actin filament assembly, alone or in combination with taxol, inhibited the cold-induced shape change but not dilation of the open canalicular system. Rewarming eliminated open canalicular system dilation and restored lentiform appearance. The results indicate that microtubule coils are the major structural elements responsible for disc shape and its restoration after submaximal stimulation or rewarming of chilled platelets.
An investigation into the mechanical behavior of microwave-cured glass–epoxy composites is carried out and the results are compared with those of thermally cured composites. Microwave curing of a ...glass fiber reinforced bifunctional epoxy resin system (LY556/HY951) is carried out in custom-built, multi-mode, industrial microwave cure equipment operating at a frequency of 2450 MHz. Through tensile, compression, and flexural strength tests, it is demonstrated that while the mechanical properties of microwave-cured composites compare well with those of the thermally cured ones, the microwave curing process, per se, results in a significant reduction in the process cycle time and power consumption.
Directed and elliptic flow of inclusive photons near mid-rapidity in 158
A GeV Pb
+
Pb collisions has been studied. The data have been obtained with the photon spectrometer LEDA of the WA98 ...experiment at the CERN SPS. The flow strength has been measured for various centralities as a function of
p
T
and rapidity over
0.18
<
p
T
<
1.5
GeV
/
c
and
2.3
<
y
<
2.9
. The angular anisotropy has been studied relative to an event plane obtained in the target fragmentation region that shows the elliptic flow to be in-plane. The elliptic flow has also been studied using two-particle correlations and shown to give similar results. A small directed flow component is observed. Both the directed and elliptic flow strengths increase with
p
T
. The photon flow results are used to estimate the corresponding neutral pion flow.
An investigation was carried out to assess the combining ability and nature of gene action in respect of juice yield and itsattributing traits in 72 new hybrids of sweet sorghum developed by crossing ...4 male sterile lines with 18 testers in Line × Testermating design and grown in Randomized Block Design with two replications during kharif 2008. The variance among the linesin respect of their general combining ability was highly significant for days to 50 per cent flowering, nodes per plant and juiceyield, where as variance among testers was significant for juice yield. The variance due to line × tester interaction was significantfor all the characters indicating differential response of lines with different testers. SCA variance was relatively higher inmagnitude for all the traits indicating predominance of non-additive gene action in the genetic control of these traits. ICSA 38and ICSA 102 among the lines and ICSV 93046, E 36-1, ICSV 700, SEREDO and GD 65008 among the testers, were identifiedas good general combiners indicating their ability in transmitting additive genes in the desirable direction to their progenies.Highly significant sca effects were observed in most of the hybrids for all the characters studied and good specific combiners fordifferent characters involved parents with high × high, high × low, low × high and low × low general combinations
Adipocyte differentiation is regulated largely through the actions of the peroxisome proliferator-activated receptor (PPAR) gamma nuclear receptor and the insulin signaling pathway. ...3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPARgamma signaling pathways, and their association with adipocyte differentiation. Coexpression of PDK1 and PPARgamma1 in 293T cells stimulated PPARgamma response element-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPARgamma1 activity was comparable in magnitude to the coactivator activated in breast cancer-1, and was blocked by either the corepressor silencing mediator of retinoid and thyroid hormone receptor or dominant-negative PAX8-PPARgamma1. Heterologous Gal4-PPARgamma1 assays indicated that PDK1 interacted with the ligand binding domain, and physically associated with PPARgamma1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPARgamma1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the alpha-helical hydrophobic motifs in PDK1, L(268)XII, and V(313)XXLL, and expression of the alpha-helical region encompassing these motifs stimulated PPARgamma response element-dependent transcription. PDK1-PPARgamma interaction was confirmed by chromatin immunoprecipitation analysis of the lipoprotein lipase and adipocyte fatty acid-binding protein promoters. In cells expressing PDK1 and PPARgamma, binding to PPARgamma response elements occurred, which was enhanced by treatment with a PPARgamma agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPARgamma agonist that was comparable to the response of PPARgamma1-transfected cells in the presence of agonist; expression of PDK1 and PPARgamma resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPARgamma agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPARgamma1 coactivator independently of its catalytic activity and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.
Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive ...cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.
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