Skin surface electric potential induced by ion-flux through epidermal cell layers

The skin surface electric potential has been widely used for psychological studies because it is sensitive to emotional conditions. We measured the electric potential on the surface of hairless mice skin in organ culture with several physiological factors. Disruption of mitochondrial function and inhibition of ATPase reduced the skin surface potential 50-70%. Calcium, potassium, and sodium channel blockers also reduced the potential. A calcium-specific and potassium ionophore reduced the potential, but the calcium and magnesium ionophore increased it. EDTA decreased the potential but EGTA had no effect. Skin surface barrier disruption reduced the potential and calcium and potassium channel blockers partially prevented the decrease. Substance P and corticotropin-releasing factor decreased the potential, and antagonists blocked the decreases. These results suggest that the ion flux in the nucleated layer of the epidermis induce the skin surface potential and it is influenced by environmental and neuroendocrinological factors.     

Synthesis of C-mannopyranosylphloroacetophenone derivatives and their anomerization

The reaction of 2,3,4-tri-O-benzyl-alpha-L-rhamnopyranosyl fluoride (6-deoxy-2,3,4-tri-O-benzyl-alpha-L-mannopyranosyl fluoride) with 2,4-dibenzylphloroacetophenone, in the presence of boron trifluoride.diethyl etherate, afforded both the 3-C-alpha-L- and the 3-C-beta-L-rhamnopyranosylphloroacetophenone derivatives. The 3-C-alpha-L-rhamnoside was produced as a major product, while the 3-C-beta-L-rhamnoside was produced as a minor product via anomerization of the 3-C-alpha-L-rhamnoside. Alternatively, the reaction of 2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl fluoride with 2,4-dibenzylphloroacetophenone afforded both the 3-C-alpha-D- and the 3-C-beta-D-mannnopyranosylphloroacetophenone derivatives under identical conditions. The 3-C-beta-D-mannoside was produced as a major product via anomerization of the 3-C-alpha-D-mannoside during the reaction. These differences in composition result apparently from the magnitude of the 1,3-diaxial interactions between the C-3 and C-5 positions in these sugar moieties.     

Cleavage of the C-C linkage between the sugar and the aglycon in C-glycosylphloroacetophenone, and the NMR spectral characteristics of the resulting di-C-glycosyl compound

The treatment of unprotected mono-C-beta-D-glucopyranosylphloroacetophenone with a cation-exchange resin in anhydrous acetonitrile afforded both a phloroacetophenone and a di-C-beta-D-glucopyranosylphloroacetophenone. Treatment of an unprotected mono-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone (mono-C-2-deoxy-beta-D-glucopyranosylphloroacetophenone) also afforded both the aglycon and di-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone. The reaction mixtures were acetylated, and the structures of the isolated products were determined by NMR spectroscopy. This is the first demonstration of the formation of a di-C-glycosyl compound during the chemical cleavage of the C-C linkage between the sugar and the aglycon in an aryl C-glycosyl derivative.     

Soybean sterol composition and utilization by Phytophthora sojae

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.     

Expression and purification of recombinant Kaposi's sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system

The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.     

Sensitive determination of pethidine in body fluids by gas chromatography-tandem mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography-tandem mass spectrometry (GC-MS/MS). Pethidine and 4'-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C(18) columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25-40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.     

cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFalpha

Gene expression of synoviocytes stimulated with tumor necrosis factor-alpha (TNFalpha) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFalpha stimulation. These findings suggest that the synoviocyte response to TNFalpha stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.     

Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris

A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes. We have designated this protein NsRpoTp.     

Inhibitory effect of quercetin on carrageenan-induced inflammation in rats

We examined the effect of quercetin on the inflammatory response induced by carrageenan in the rat. Air pouches were induced subcutaneously on the backs of rats and injected with carrageenan. The rats were treated with either vehicle or quercetin at a dose of 10 mg/kg one hour before carrageenan challenge. Fourty-eight hour after carrageenan challenge, the air pouches were removed and analyzed. The volume, protein amounts and cell counts in the exudation obtained from the quercetin-treated animals were significantly reduced compared to those from vehicle-treated animals. The contents of PGE(2), TNF-alpha, RANTES, MIP-2 and the mRNA for cyclooxygenase-2 were also suppressed in these rats. The histological examination displayed the suppression of the inflammatory response in the pouch tissues from quercetin-treated rats. As the anti-inflammatory effect of the flavonols was more or less at the similar level among the quercetin-, isoquercitrin- or rutin-treated rats, it appeared that the sugar parts did not influence on the anti-inflammatory effect.Our study indicated that the flavonols modulated the inflammatory response, at least in part, by modulating the prostanoid synthesis as well as cytokine production.     

Collagen XXIV,a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.