Syntheses of sphingosine-1-phosphate stereoisomers and analogues and their interaction with EDG receptors

Sphingosine-1-phosphate (S1P) is considered to be an important regulator of diverse biological processes acting as a natural ligand to EDG receptors. As a preliminary study to develop potent and selective agonist and antagonist for EDG receptors, we report synthesis of S1P stereoisomers and analogues and their binding affinities to EDG-1, -3, and -5.     

RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.     

Kaposi's sarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cells to induce inflammatory cytokine production

Kaposi's sarcoma is an inflammatory cytokine-mediated angioproliferative disease which is triggered by infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV contains an open reading frame, K14, that has significant homology with cellular OX2, designated viral OX2 (vOX2). In this report, we demonstrate that vOX2 encodes a glycosylated cell surface protein with an apparent molecular mass of 55 kDa. Purified glycosylated vOX2 protein dramatically stimulated primary monocytes, macrophages, and dendritic cells to produce the inflammatory cytokines interleukin 1beta (IL-1beta), IL-6, monocyte chemoattractant protein 1, and TNF-alpha. Furthermore, expression of vOX2 on B lymphocytes stimulated monocytes to produce inflammatory cytokines in mixed culture. These results demonstrate that like its cellular counterpart, vOX2 targets myeloid-lineage cells, but unlike cellular OX2, which delivers a restrictive signal, KSHV vOX2 provides an activating signal, resulting in the production of inflammatory cytokines. Thus, this is a novel viral strategy where KSHV has acquired the cellular OX2 gene to induce inflammatory cytokine production, which potentially promotes the cytokine-mediated angiogenic proliferation of KSHV-infected cells.     

Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls

The labeling of targeting peptides with (99m)Tc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac-[(99m)Tc(CO)(3)](+) core for introducing (99m)Tc in biomolecules in general and in peptides in particular. The three coordination sites available in [(99m)Tc(OH(2))(3)(CO)(3)](+) can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed-ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.     

Efficacy of β-mannanase supplementation to corn–soya bean meal-based diets on growth performance,nutrient digestibility,blood urea nitrogen,faecal coliform and lactic acid bacteria and faecal noxious gas emission in growing pigs

A study was conducted to determine the efficacy of β-mannanase supplementation to a diet based on corn and soya bean meal (SBM) on growth performance, nutrient digestibility, blood urea nitrogen (BUN), faecal coliforms and lactic acid bacteria, and noxious gas emission in growing pigs. A total of 140 pigs [(Landrace × Yorkshire) × Duroc; average body weight 25 ± 3 kg] were randomly allotted to a 2 × 2 factorial arrangement with dietary treatments consisting of hulled or dehulled SBM without or with supplementation of 400 U β-mannanase/kg. During the 6 weeks of experimental feeding, β-mannanase supplementation had no effect on body weight gain, feed intake and gain:feed (G:F) ratio. Compared with dehulled SBM, feeding hulled SBM caused an increased feed intake of pigs in the entire trial (p = 0.05). The G:F ratio was improved in pigs receiving dehulled SBM (p < 0.05). Dietary treatments did not influence the total tract digestibility of dry matter, nitrogen and gross energy. Enzyme supplementation reduced (p < 0.05) the population of faecal coliforms and tended to reduce the NH₃ concentration after 24 h of fermentation in a closed box containing faecal slurry. Feeding hulled SBM tended to reduce NH₃ emission on days 3 and 5 of fermentation. In conclusion, mannanase supplementation had no influence on growth performance and nutrient digestibility but showed a positive effect on reducing coliform population and tended to reduce NH₃ emission. Dehulled SBM increased G:F ratio and hulled SBM tended to reduce NH₃ emission.     

Long-Term Clinical Outcome of Internal Globus Pallidus Deep Brain Stimulation for Dystonia

Background

GPi (Internal globus pallidus) DBS (deep brain stimulation) is recognized as a safe, reliable, reversible and adjustable treatment in patients with medically refractory dystonia.

Objectives

This report describes the long-term clinical outcome of 36 patients implanted with GPi DBS at the Neurosurgery Department of Seoul National University Hospital.

Methods

Nine patients with a known genetic cause, 12 patients with acquired dystonia, and 15 patients with isolated dystonia without a known genetic cause were included. When categorized by phenomenology, 29 patients had generalized, 5 patients had segmental, and 2 patients had multifocal dystonia. Patients were assessed preoperatively and at defined follow-up examinations postoperatively, using the Burke-Fahn-Marsden dystonia rating scale (BFMDRS) for movement and functional disability assessment. The mean follow-up duration was 47 months (range, 12–84)

Results

The mean movement scores significantly decreased from 44.88 points preoperatively to 26.45 points at 60-month follow up (N = 19, P = 0.006). The mean disability score was also decreased over time, from 11.54 points preoperatively to 8.26 points at 60-month follow up, despite no statistical significance (N = 19, P = 0.073). When analyzed the movement and disability improvement rates at 12-month follow up point, no significant difference was noted according to etiology, disease duration, age at surgery, age of onset, and phenomenology. However, the patients with DYT-1 dystonia and isolated dystonia without a known genetic cause showed marked improvement.

Conclusions

GPi DBS is a safe and efficient therapeutic method for treatment of dystonia patients to improve both movement and disability. However, this study has some limitations caused by the retrospective design with small sample size in a single-center.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.