Overexpression of SR proteins and splice variants modulates chondrogenesis

Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations.     

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.     

Hormone-like fibroblast growth factors and metabolic regulation

The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called “hormone-like” subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.     

Genotype analysis of the CYP2C19 gene in HCV-seropositive patients with cirrhosis and hepatocellular carcinoma

This study was conducted to assess whether the genotypic frequency of Smephenytoin 4'-hydroxylase CYP2C19 gene differs in Japanese cirrhotic patients who developed hepatocellular carcinoma. Thirty-eight patients with cirrhosis were studied. The wild-type allele CYP2C19*1 and the two mutated alleles, CYP2C19*2 and CYP2C19*3, were identified by PCR-RFLP method. Individuals with homozygous CYP2C19*2 or CYP2C19*3 mutation and those with CYP2C19*2 and CYP2C19*3 heterozygous mutation were predicted to be the poor metabolizer (PM) phenotype. The overall frequency of PM predicted from the genotyping analysis was 29% (11 of the 38 patients), consisting of 5 patients homozygous for CYP2C19*2, two homozygous for CYP2C19*3 and four heterozygous for the two defects. Among 24 HCV-seropositive patients with cirrhosis and hepatocellular carcinoma, the frequency of PM was 41.7% and significantly higher than that observed in 186 healthy controls. We postulate that the PM phenotype caused by the mutation of CYP2C19 gene in cirrhotic patients with HCV infection is associated with a high risk for developing hepatocellular carcinoma.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Validation of a Laboratory Method for Evaluating Dynamic Properties of Reconstructed Equine Racetrack Surfaces

Background

Racetrack surface is a risk factor for racehorse injuries and fatalities. Current research indicates that race surface mechanical properties may be influenced by material composition, moisture content, temperature, and maintenance. Race surface mechanical testing in a controlled laboratory setting would allow for objective evaluation of dynamic properties of surface and factors that affect surface behavior.

Objective

To develop a method for reconstruction of race surfaces in the laboratory and validate the method by comparison with racetrack measurements of dynamic surface properties.

Methods

Track-testing device (TTD) impact tests were conducted to simulate equine hoof impact on dirt and synthetic race surfaces; tests were performed both in situ (racetrack) and using laboratory reconstructions of harvested surface materials. Clegg Hammer in situ measurements were used to guide surface reconstruction in the laboratory. Dynamic surface properties were compared between in situ and laboratory settings. Relationships between racetrack TTD and Clegg Hammer measurements were analyzed using stepwise multiple linear regression.

Results

Most dynamic surface property setting differences (racetrack-laboratory) were small relative to surface material type differences (dirt-synthetic). Clegg Hammer measurements were more strongly correlated with TTD measurements on the synthetic surface than the dirt surface. On the dirt surface, Clegg Hammer decelerations were negatively correlated with TTD forces.

Conclusions

Laboratory reconstruction of racetrack surfaces guided by Clegg Hammer measurements yielded TTD impact measurements similar to in situ values. The negative correlation between TTD and Clegg Hammer measurements confirms the importance of instrument mass when drawing conclusions from testing results. Lighter impact devices may be less appropriate for assessing dynamic surface properties compared to testing equipment designed to simulate hoof impact (TTD).

Potential Relevance

Dynamic impact properties of race surfaces can be evaluated in a laboratory setting, allowing for further study of factors affecting surface behavior under controlled conditions.     

Effects of immunosuppressive therapy on murine Leishmania infantum visceral leishmaniosis

We evaluated the effect of immunosuppressive therapy on the course of infection, the spleen cell immunophenotype and cytokine production during murine Leishmania infantum visceral leishmaniosis (VL). Rousseau et al. [1] recently reported that prolonged administration of dexamethasone induces limited reactivation of chronic murine visceral leishmaniosis, with no clear Th1-Th2 cytokine patterns. We found that another glucocorticoid, hydrocortisone acetate, had similar effects during acute visceral leishmaniosis, i.e. an increase in parasite burden in the spleen, but not the liver, of infected mice. A significant increase in parasite burden in both the liver and the spleen was only achieved when mice were treated with combined dexamethasone + pentoxifylline immunotherapy; increases in parasite burden were never associated with a specific spleen cell immunophenotype or a Th1-Th2 cytokine secretion profile.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.