Glycosylation is essential for efficient secretion but not for permeability-enhancing activity of vascular permeability factor (vascular endothelial growth factor)

The hyperpermeability of the microvasculature supplying solid tumors is largely attributable to a heterodimeric Mr 34,000-43,000 tumor-secreted protein, vascular permeability factor. Upon reduction, the vascular permeability factor secreted by line 10 tumor cells is resolved by SDS-PAGE into 3 discrete bands of Mr 24,000, 19,500, and 15,000. We demonstrate here that line 10 vascular permeability factor is an N-linked glycoprotein. Nonglycosylated vascular permeability factor migrates on reduced SDS-PAGE as two bands of Mr 20,000 and 15,000. Pulse-chase studies demonstrated that all three chains of native vascular permeability factor were secreted rapidly following synthesis and at equal rates, with a cellular half-retention time of approximately 37 min. When glycosylation was prevented by tunicamycin, individual bands of nonglycosylated vascular permeability factor were also secreted at equivalent rates, but much more slowly (approximately 60 min) than native glycoprotein. Both glycosylated and nonglycosylated forms of vascular permeability factor were equally potent at increasing dermal vessel permeability.     

Changes of the oxidative phosphorylation in mitchondria of rat skeletal muscle following strenous exercise.

The rate of oxygen consumption of mitochondria from rat muscles at pH 7.4 is elevated by 1-lactate. The respiratory control ratio and the ADP/O-ratio are decreased under these conditions. Acidification to pH 6.5 in the absence of 1-lactate does not change the interpreted mitochondrial functions. The experimental data are discussed as a partial uncoupling effect of 1-lactate on the oxidative phosphorylation. Similar changes in those mitochondrial functions are found after short-time intensive swimming exercise of rats. These variations might be a reason for the sometimes described reduced aerobic performance after intensive work.     

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ～22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.     

Peptide-Guided Surface-Enhanced Raman Scattering Probes for Localized Cell Composition Analysis

The ability to control the localization of surface-enhanced Raman scattering (SERS) nanoparticle probes in bacterial cells is critical to the development of analytical techniques that can nondestructively determine cell composition and phenotype. Here, selective localization of SERS probes was achieved at the outer bacterial membrane by using silver nanoparticles functionalized with synthetic hydrophobic peptides.     

Impact of cefotaxime on somatic embryogenesis and shoot regeneration in sugarcane

A cephalosporin antibiotic, cefotaxime (Omnatax™) promoted somatic embryogenesis and subsequent shoot regeneration in vitro from spindle in sugarcane irrespective of the genotypes as (CoJ 83, CoJ 88 and CoJ 64) culturered on MS medium with 2,4-D (2.5 mgl⁻¹) and kinetin (0.5 mgl⁻¹). Seven different concentrations of cefotaxime (100, 200, 300, 400, 500, 600 and 700 mgl⁻¹) were tested to find the optimal concentration of cefotaxime for somatic embryogenesis from callus cultures. Among the three varieties, calli of variety CoJ 83 incubated on MS medium with 2,4-D (2.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) + cefotaxime (500 mgl⁻¹) exhibited maximum somatic embryogenesis. To improve shoot regeneration, the callus was transferred to MS medium with BAP (0.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) in combination with different levels of cefotaxime. Highest frequency of shoot regeneration was observed in callus of CoJ 83 in the presence of 500 mgl⁻¹ cefotaxime. The plantlets could be successfully hardened in polybags and transferred to soil, where they exhibited normal growth. Our results convincingly demonstrated that cefotaxime improves somatic embryogenesis from spindle and regeneration from embryogenic calli of sugarcane and hence can be strongly recommended for rapid and large scale multiplication of sugarcane.Key words: Saccharum officinarum L., leaf segments, callus, plant regeneration, antibiotic     

Structure–activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor

Structure–activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund’s adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res. 1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain 2006, 10, 537].     

minifly, a Drosophila gene required for ribosome biogenesis

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.     

Modeling of the detachment of a molecule from a surface: illustration of the "Bell-Evans effect"

This article deals with the modeling of the detachment of a molecule initially adsorbed on a surface and submitted to an external force whose strength increases with time. By means of an atomic force microscope (AFM), it is possible to measure the force when the molecule separates from the substrate. However, it is known that this force depends to a large extend on the rate at which the pulling force is applied ("Bell-Evans effect"). Two models are described to illustrate this behavior. First, a random walk approach is suggested to reveal the fundamental principle of the escape over a time-dependent energy barrier. Second, a multi bead-and-spring model is proposed to mimic the AFM experiment and numerical simulations, based on Brownian dynamics, are performed.     

Collagen I initiates endothelial cell morphogenesis by inducing actin polymerization through suppression of cyclic AMP and protein kinase A

Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha(1)beta(1) and alpha(2)beta(1), followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords.