Porous composites of hydroxyapatite‐filled poly[ethylene‐co‐(vinyl acetate)] for tissue engineering

A novel porous composite of hydroxyapatite/poly[ethylene‐co‐vinyl acetate)] (HAP/EVA) having better osteointegration was fabricated by gas foaming technique using a non toxic gas blowing agent intended for bone replacement applications. Combined techniques of scanning electronic microscopy (SEM) and X‐ray microcomputed tomography (µCT) analysis showed that the pore size and pore volume of the porous composite decrease with the increase of HAP content. The gravimetric analysis evidenced for good pore interconnectivity within the porous composites. Energy dispersive X‐ray analysis (EDX) studies inveterated the even scattering of Ca ions which in turn indicate the uniform dispersion of HAP particles in the composites. The significant gradation in Ca ion concentration seen in EDX studies is well accordance with the amount of HAP loading in the sample. Mechanical properties of the porous composite having different HAP content were measured to have the compressive strength varying from 1.06 to 2.2 MPa. Non‐cytotoxic character of the material was observed by the cytocompatibility studies. The metabolic activity of L929 cells seeded on the material assessed by [3‐(4,5‐dimethylthiazol)‐2‐yl]‐2,5‐diphenyltertrazolium bromide (MTT) assay was found to be 91.8%. The adhesion and migration of the cells inside the pore walls were visualized by confocal microscopy. Copyright © 2010 Society of Chemical Industry     

Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers

Values of skeletal muscle intracellular PO2 during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular PO2 in isolated single skeletal muscle fibers by using O2-dependent quenching of a phosphorescent-O2 probe is described. Intact single skeletal muscle fibers from Xenopus laevis were dissected from the lumbrical muscle and mounted in a glass chamber containing Ringer solution at 20 degreesC. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium-meso-tetra (4-carboxyphenyl) porphine bound to bovine serum albumin was injected into single fibers by micropipette pressure injection. Phosphorescence-decay curves (average of 10 rapid flashes) were recorded every 7 s from single cells (n = 24) in which respiration had been eliminated with NaCN, while the PO2 of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculated at the varied extracellular PO2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 microseconds at an extracellular PO2 of 159 Torr to 650-700 microseconds at 0 Torr. The phosphorescent lifetimes for the varied PO2 were used to calculate, by using the Stern-Volmer relationship, the phosphorescence-quenching constant (100 Torr-1. s-1), and the phosphorescence lifetime in a zero-O2 environment (690 microseconds) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular PO2 in isolated single skeletal muscle fibers.     

High-frequency retrotransposition of a marked I factor in Drosophila melanogaster correlates with a dynamic expression pattern of the ORF1 protein in the cytoplasm of oocytes

To study the expression of the I factor, a non-long-terminal-repeat retrotransposon responsible for I-R hybrid dysgenesis in Drosophila melanogaster, we have tagged the ORF1 protein (ORF1p) by inserting the HA epitope in its N-terminal region. In transgenic flies, this modification is compatible with a high rate of autonomous transposition and allows direct estimation of the transposition frequency. I factor transposes in the germline of females (SF) that are daughters from crosses between I strain males (which contain active copies of the I factor) and R strain females (which do not). We analyzed the expression pattern of ORF1p by indirect immunofluorescence. Its expression correlates with retrotransposition. During oogenesis ORF1p appears unexpectedly as a cytoplasmic product, which accumulates with a specific pattern into the oocyte. A comparison of the expression patterns under conditions that modify the transposing activity of the element clarifies some aspects of I-factor functioning in the transposition process.     

Regulation of the expression of peripheral benzodiazepine receptors and their endogenous ligands during rat sciatic nerve degeneration and regeneration: a role for PBR in neurosteroidogenesis

This study addresses (1) the relationship between headache presence/intensity at time of testing and neurocognitive performance, and (2) the probability that testing triggers or intensifies pain. Subjects were 125 patients with chronic posttraumatic headache (mean = 2.67 years post injury) who completed a 4-hour test battery emphasizing memory. Comparisons of 34 individual tests/subtests and the five Wechsler Memory Scale-Revised (WMS-R) indices of relative memory impairment for 73 patients with no headache or mild headache versus 52 patients with moderate to severe pain revealed no significant differences. Testing intensified existing headaches for 55% but triggered headache for only 1 of 20 (5%; P =.00003). Results support the validity of neuropsychological test performance regardless of pain level, although testing can be painful.     

Relationship between plasma phospholipid transfer protein activity and HDL subclasses among patients with low HDL and cardiovascular disease

NK lysin is a 9-kDa polypeptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. It is produced by cytolytic lymphocytes and is cytolytic against a number of different types of tumor cells. Here we report the binding of NK lysin to lipopolysaccharide (LPS) and its anti-LPS activity. NK lysin binds to matrix-coated LPS from Escherichia coli, Pseudomonas aeruginosa, and different strains of Salmonella enterica. Lipid A and polymyxin B inhibited the binding, demonstrating a preferential interaction of NK lysin with the lipid part of LPS. Chromium-labeled lymphoma cells were lysed by NK lysin, and LPS dose-dependently inhibited the cytolysis at equimolar amounts. In the same manner, NK lysin inhibited certain LPS-stimulated effects on mouse bone marrow cells as well as LPS binding to mouse granulocytes. These results suggest that NK lysin may be a another natural LPS-binding protein from lymphocytes that may participate in the endogenous defense response associated with elevated concentrations of LPS.     

Prevention of lens damage associated with cigarette smoke exposure in rats by alpha-tocopherol (vitamin E) treatment

PURPOSE: To evaluate the possible protective effect and mechanism of alpha-tocopherol (vitamin E) treatment on lens degeneration associated with in vivo exposure to cigarette smoke and to further clarify the role of iron in cigarette smoke-generated lens damage. METHODS: Twenty-eight male Wistar rats were randomly divided into four equal groups. Rats in groups 3 and 4 were exposed to cigarette smoke for 1 hour each day over 90 consecutive days, and rats in groups 1 and 2 were treated in similar fashion but only exposed to room air. Additionally, vitamin E was given to the rats in groups 2 and 4 via intramuscular route. At the end of the study, both eyes of all the animals were enucleated; one eye was prepared for histopathologic examination, and the fellow eye was used for the measurement of iron and calcium levels. RESULTS: Significantly higher iron and calcium levels were observed in the lenses of group 3 rats than in other groups. Similar comparisons performed between groups 1 and 2, groups 1 and 4, and groups 2 and 4 did not show any significant difference. Distinct histopathologic changes in the anterior lens epithelium, such as hyperplasia, hypertrophy, epithelial multilayering, and the presence of epithelial cells over posterior lens capsule, observed in group 3 rats were not present in other groups. CONCLUSIONS: Cataractogenesis after cigarette smoke exposure was associated with an accumulation of iron and calcium in the rat lens, and vitamin E supplementation protected such accumulations and cataractogenesis.     

Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes

We have overexpressed and purified human dual-specificity phosphatase cdc25C from a prokaryotic expression system at high levels and in a soluble, active form, and have studied and quantified its potential to interact with cdks, cyclins and preformed cdk-cyclin complexes by fluorescence spectroscopy and size-exclusion chromatography. Our data indicate that human cdc25C forms stable complexes, through hydrophobic contacts, with cdk and cyclin monomers, as well as with preformed cdk-cyclin complexes. In vitro, cdc25C interacts with cyclin monomers with high affinity, with tenfold less affinity with cdks, and with intermediate affinity with cdk-cyclin complexes. Moreover, changes observed in the intrinsic fluorescence of cdks, cyclins and cdk-cyclin complexes upon interaction with cdc25C are indicative of concomitant conformational changes within cdks and cyclins. From our results, we propose that in vitro, in the presence of monomeric cdks and cyclins, cdc25C forms stable ternary complexes, first through a high affinity interaction with a cyclin, which may then help target cdc25C towards a cdk. We discuss the biological relevance of our results and propose that a similar, two-step mechanism of interaction between cdc25C and cdk-cyclin complexes may occur in vivo.     

A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans

The glycosylation of the equine interhaemal barrier and areola was studied throughout the period of gestation. Placentae of 35, 37, 50, 119, 152, 200, 280 and 300 days gestation were investigated, using semithin plastic embedded sections and a panel of 15 biotinylated lectins with an avidin-peroxidase revealing system. Glycosylation of the trophoblast and maternal epithelium showed the most change during the first 50 days of gestation, being associated with the initial stages of adhesion and attachment. In the trophoblast, non-bisected tri/tetraantennary complex N-glycan was only evident after day 37 and terminal N-acetyl galactosamine, alpha2,3- and alpha2,6-linked sialic acids disappeared at the same time. The areolar trophoblast exhibited some differences from microcotyledonary areas, especially with respect to 2-deoxy, 2-acetamido alpha-galactose and tri/tetraantennary, non-bisected complex N-glycan, suggesting that the differences in function between microcotyledonary and areolar trophoblast are reflected at both the morphological and the biochemical level. Granules of the maternal uterine epithelium bound many lectins, particularly those with specificity for bisected and non-bisected bi/triantennary N-linked glycan, 2-deoxy, 2-acetamido alpha-galactosyl, beta-galactosyl and some fucosylated termini. Binding to sialic acids in alpha2,3- and alpha2,6-linkage was sparse. Maternal and fetal capillaries showed little change in glycan expression over the period studied, being rich in bisected and non-bisected bi/triantennary N-linked glycan and sialic acids, with some terminal N-acetyl galactosamine and no detectable terminal fucosyl residues.     

DISPLAY LIFE OF FRESH BEEF CONTAINING DIFFERENT LEVELS OF VITAMIN E AND INITIAL MICROBIAL CONTAMINATION

Strip loins from beef cattle fed diets supplemented or not supplemented with α-tocopheryl acetate (vitamin E; 500 IU·animal⁻¹·d^-1) were fabricated into steaks inoculated at three initial levels (1.7 to 1.9; 2.3 to 2.5; 6.4 to 7.1 log CFU/cm²) of bacterial contamination (from over aged retail steaks) and evaluated for psychrotrophic plate counts (PPC), overall appearance and color during 6 days of simulated retail display (0 to 4C). Initial contamination affected changes in PPC (P < 0.05) during display, while the highest level of initial contamination eliminated benefits of high vitamin E concentrations on product color within 4 days of retail display. However, increased vitamin E concentrations in steaks were associated with higher (P < 0.05) CIE a* values appearance scores when the level of initial contamination was less than 2.5 CFU/cm². Results indicated that vitamin E supplementation does not mask high levels of bacterial contamination.