3-O-Laurylglyceryl ascorbate reinforces skin barrier function through not only the reduction of oxidative stress but also the activation of ceramide synthesis.

OBJECTIVE: A higher trans-epidermal water loss (TEWL) occurs in rough skin, in elder skin and also in atopic dermatitis. An impaired skin barrier function is considered to be caused by an incomplete construction of the intercellular lamellar structure due to the quantitative reduction of ceramides. Since these symptoms coexist with oxidative stress, we hypothesized that impairment of the skin barrier function is accelerated by oxidative stress. Thus, the purpose of this study was to clarify the effect of oxidative stress on ceramide synthesis and to characterize whether antioxidants can improve skin barrier function. 3-O-Laurylglyceryl ascorbate (VC-3LG), which is a newly amphipathic derivative of ascorbic acid, was evaluated as a candidate antioxidant. METHODS: We characterized the mRNA expression levels of serine palmitoyltransferase (SPT) in normal human epidermal keratinocytes (NHEKs) treated with H2 O2 using real-time PCR analysis. In order to evaluate the effect of VC-3LG on skin barrier function, we used several assays with reconstructed human epidermis equivalents (RHEEs). RESULTS: Ceramide synthesis was down-regulated in NHEKs by oxidative stress. Treatment with VC-3LG abrogated the down-regulation of SPT mRNA in NHEKs caused by oxidative stress, and stimulated SPT mRNA expression levels. In experiments characterizing the antioxidative properties of VC-3LG, VC-3LG reduced oxidative stress in NHEKs by up-regulating catalase mRNA expression. In addition, VC-3LG stimulated the skin barrier function in RHEEs, which had lower TEWL values compared with untreated RHEEs. Furthermore, VC-3LG increased the quantity of ceramide in RHEEs. CONCLUSION: Taken together, we conclude that VC-3LG reinforces the skin barrier function due to its reduction of oxidative stress and its promotion of ceramide synthesis.

Involvement of gicerin in the extension of microvilli.

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.

Synthesis of a nitrogen analogue of salacinol and its alpha-glucosidase inhibitory activity.

A nitrogen analogue 4 of the naturally occurring sulfonium ion salacinol (1), a potent alpha-glucosidase inhibitor isolated from the Ayruvedic medicine Salacia reticulata, was synthesized and its inhibitory activity against alpha-glucosidase tested. Substitution of the sulfur atom in 1 with a nitrogen reduced the activity considerably. The solid-state stereostructure of the related compound (5) was determined on the basis of single crystal X-ray measurement.

Absolute stereostructures of novel norcadinane- and trinoreudesmane-type sesquiterpenes with nitric oxide production inhibitory activity from Alpinia oxyphylla.

Novel 14-norcadinane-type sesquiterpenes, oxyphyllenodiols A and B, and 11,12,13-trinoreudesmane-type sesquiterpenes, oxyphyllenones A and B, were isolated from the methanolic extract of kernels of Alpinia oxyphylla. The absolute stereostructures of these norsesquiterpenes were determined on the basis of physicochemical and chemical evidence. In addition, oxyphyllenodiol A and oxyphyllenone A were found to inhibit the NO production in lipopolysaccharide-activated macrophages.

Kheper, a novel ZFH/deltaEF1 family member, regulates the development of the neuroectoderm of zebrafish (Danio rerio).

Kheper is a novel member of the ZFH (zinc-finger and homeodomain protein)/deltaEF1 family in zebrafish. kheper transcripts are first detected in the epiblast of the dorsal blastoderm margin at the early gastrula stage and kheper is expressed in nearly all the neuroectoderm at later stages. kheper expression was expanded in noggin RNA-injected embryos and also in swirl mutant embryos and was reduced in bmp4 RNA-injected embryos and chordino mutant embryos, suggesting that kheper acts downstream of the neural inducers Noggin and Chordino. Overexpression of Kheper elicited ectopic expansion of the neuroectoderm-specific genes fkd3, hoxa-1, and eng3, and the ectopic expression of hoxa-1 was not inhibited by BMP4 overexpression. Kheper interacted with the transcriptional corepressors CtBP1 and CtBP2. Overexpression of a Kheper mutant lacking the homeodomain or of a VP16-Kheper fusion protein disturbed the development of the neuroectoderm and head structures. These data underscore the role of Kheper in the development of the neuroectoderm and indicate that Kheper acts as a transcriptional repressor.

Percutaneous penetration of ozagrel and the enhancement produced by saturated fatty acids.

The effects of a series of fatty acids on the percutaneous penetration of ozagrel (OZ), a selective thromboxane A2 synthetase inhibitor, through rat skin and the mechanism by which fatty acids enhance the skin penetration of OZ were examined in vitro. Lauric acid, at the fatty acid: OZ molar ratio of 2 : 1, was the most potent agent as far as increasing the skin penetration was concerned, with a flux 24-fold higher than that without fatty acid. A molar ratio of 3 : 1 also produced a large enhancing effect, comparable with that of a molar ratio of 2 : 1. When the gel formulation with lauric acid (molar ratio of 2 : 1) was applied to the skin for 6 h, the amount of drug penetrating into the skin was significantly increased compared with that after the formulations without lauric acid and with capric and palmitic acids. However, lauric acid did not change the apparent partition coefficient of OZ between n-heptane and phosphate buffer (pH 7.4). The 13C-NMR spectra of OZ was also unaffected by the addition of lauric acid, indicating that a complex or ion pair with lauric acid was not formed. A possible mechanism for the enhancing effect is the increased incorporation of lauric acid with OZ into the bulk lipid phase of the stratum corneum, where the fatty acid would act as a co-penetrant enhancing passage through the stratum corneum.

Inhibition by naloxone of promoter activity of the neurofilament gene in SK-N-SH cells.

Chronic administration of morphine is known to decrease the levels of neurofilaments (NFs) in the ventral tegmental area. We ligated a promoter region of the mouse 68-KDa neurofilament (NF-68) gene to the pGL3-enhancer vector containing a luciferase gene, transfected it into SK-N-SH cells and then analyzed transcriptional activity in the cells treated with agonists or antagonists of opiate receptors. The activity of the NF-68 promoter was suppressed by naloxone about 55% at 10(-5) M and 30% at 10(-7) M at 48 h, but suppressed not by morphine. Naltrexone at 10(-5) M suppressed the promoter activity about 20%, but levallorphan, DAMGO, DPDPE and U50488 did not. The inhibition by naloxone was dose-dependent and not reversed by morphine. The inhibitory effect of naloxone was not observed in N18TG-2 cells and PC12 cells. Experiments with various deletion mutants revealed that a region responsible for naloxone suppression spans from -328 to -101 in the gene. These results suggest that naloxone has the ability to suppress transcriptional activity in some neurons.

Synthesis of 2,3-dihydroinosine derivatives by reduction using BH3-THF.

A novel reductive method for the chemical modification of nucleosides is described. Reaction of inosine derivatives with boran-THF resulted in the regioselective reduction of purine ring to afford the corresponding 2,3-dihydroinosine derivatives in moderate yields.

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule.

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.

Enhancement of the oral bioavailability of phenytoin by N-acetylation and absorptive characteristics.

To improve the absorbability of phenytoin (DPH), a prodrug, N-acetyl-DPH (EDPH), was synthesized, and the absorptive characteristics and pharmacokinetics of the prodrug were evaluated in rats. EDPH was rapidly hydrolyzed to DPH in the intestinal fluid and the mucosa (rate constant, 0.055 and 0.169 min(-1), respectively). The plasma concentrations of DPH after intravenous dosing of EDPH declined in a biexponential manner, although two different elimination patterns were observed in these rats. When dosed orally (25 mg/kg, DPH equivalent), the plasma levels of DPH converted from the prodrug were significantly higher and more sustained than those after DPH alone, giving bioavailability 11.4 (rapid decay) and 9.1 times (slow decay) as high, respectively, as that after DPH alone. The concentrations of DPH distributed into the mucosa of the duodenum and jejunum 1 and 5 h after oral dosing of EDPH were significantly higher than those after DPH alone. The prodrug and DPH converted from the prodrug dissolved 2-4 fold more than DPH alone in bile salt solution and bile salt-oleic acid mixed micelles, indicating the increased solubility of the prodrug in the intestinal fluid. It is concluded from the data that such high solubility of EDPH enhanced the intestinal absorption of the prodrug, part of which would be absorbed in the amide form, and thus gave the high bioavailability.

A cytotoxic nonstructural protein, NS1, of human parvovirus B19 induces activation of interleukin-6 gene expression.

We examined the biological function of a nonstructural regulatory protein, NS1, of human parvovirus B19. Because of the cytotoxic activity of NS1, human hematopoietic cell lines, K562, Raji, and THP-1, were established as transfectants which produce the viral NS1 protein upon induction by using bacterial lactose repressor/operator system. NS1 was significantly produced in the three transfectant cells in an inducer dose- and time-dependent manner. Surprisingly, these three transfectants secreted an inflammatory cytokine, interleukin-6 (IL-6), in response to induction. However, no production of other related cytokines, IL-1beta, IL-8, or tumor necrosis factor alpha, was seen. Moreover, NS1-primed IL-6 induction was transiently demonstrated in primary human endothelial cells. Analysis with luciferase reporter plasmids carrying IL-6 promoter mutant fragments demonstrated that NS1 effect is mediated by a NF-kappaB binding site in the IL-6 promoter region, strongly implying that NS1 functions as a trans-acting transcriptional activator on the IL-6 promoter. Our novel finding, IL-6 induction by NS1, supports the possible relationship between parvovirus B19 infection and polyclonal activation of B cells in rheumatoid arthritis and indicates that NS1 protein may play a significant role in the pathogenesis of some B19-associated diseases by modulating the expression of host cellular genes.

Genomic structure of human BST-1.

BST-1 is an ectoenzyme expressed on human bone marrow Stromal cells and myeloid lineage cells, having both ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase activities. In mouse, BST-1 is also expressed on lymphoid progenitors. We isolated chromosomal DNA segments of the human BST-1 gene. The human BST-1 gene consisted of nine exons and eight introns. The length of each exon was very similar to that of the Aplysia ADP-ribosyl cyclase gene. The flanking region of the BST-1 gene contained several potential binding sites for nuclear factors, NF-kappa B, p53, NF-IL6, CREB, PEA3, E2A, C/EBP, AP3, AP2 and SP1 and consensus sequences for gamma-IRE and ISRE like element.

Pharmacokinetics of indomethacin ester prodrugs: gastrointestinal and hepatic toxicity and the hydrolytic capacity of various tissues in rats.

In order to develop a potential prodrug of indomethacin (IM) which causes less irritation to the gastrointestinal mucosa, the ester prodrugs [butyl ester (IM-BE) and octyl ester (IM-OE)] of IM were synthesized and evaluated for their ulcerogenic activity and hepatic injury after oral administration in rats. Additionally, the kinetics of hydrolysis of the prodrugs were examined to characterize the tissues or organs capable of hydrolyzing the ester bonds. The plasma levels of IM after the oral administration of IM-OE and IM-BE were comparatively low compared with those after IM, with a small bioavailability (2.1 and 15.0%, respectively). Ulcerogenic activity and hepatic injury, expressed by decreased hepatic microsomal enzyme activities, were hardly seen after repeated oral administration of the prodrugs, in contrast with the severely irritating effects of IM alone. Hydrolysis of the prodrugs was adequately described by first-order kinetics. IM-BE was relatively rapidly hydrolyzed in plasma, skin and whole blood, but the hydrolysis in the intestinal mucosa and liver was very slow. The hydrolytic rates for IM-OE were exceedingly small or negligible. These results indicate that the main part of IM-BE and IM-OE administered orally might not be hydrolyzed to IM in the gastrointestinal tract, and that the ester prodrugs themselves were absorbed through the mucosa; also, that the hydrolysis of ester bonds would be carried out mainly in the circulatory system. Consequently, IM-BE seems to be an ideal prodrug of IM.

Zebrafish elav/HuC homologue as a very early neuronal marker.

Drosophila ELAV, a neuron-specific RNA binding protein, is expressed in all neurons right after their birth. This specific pattern of expression has led to its use as a pan-neuronal marker. At least three members of the elav family, HuD, HuC/ple21 and Hel-N1, have been reported to be neuron-specific in vertebrates, although it is unknown which member of this family is expressed at the time of early neuronal determination. We have isolated a zebrafish elav/HuC homologue (zHuC) which has 89% homology to human HuC protein. It is first expressed in the neuronal precursor cells in the neural plate immediately after gastrulation, and then high expression levels persist in most regions of the nervous system. HuC, like elav in Drosophila, may be one of the earliest neuronal markers in zebrafish.

Stage-specific expression of mouse BST-1/BP-3 on the early B and T cell progenitors prior to gene rearrangement of antigen receptor.

Human bone marrow stromal cell antigen 1 (BST-1) was identified as a glycosylphosphatidyl-inositol-anchored ectoenzyme expressed on bone marrow stromal or synovial cell lines and having the ability to facilitate pre-B cell line growth. The analysis of the expression of mouse BST-1/BP-3 on the surface of lymphoid cells in the bone marrow and thymus revealed that it was very transiently expressed on both B and T cell progenitors undergoing gene rearrangement of the antigen receptor. Among CD45R+ CD43+ B cell progenitors in the bone marrow, BST-1 expression appeared on the CD24 (heat stable antigen)+, CD19+ or CD117 (c-kit)+ population. In the thymus, BST-1 was expressed on CD4-CD8-CD3- [triple negative (TN)] CD90 (Thy-1)+ cells. In TN thymocytes, the majority of CD25+ cells and CD44(10)/- cells expressed BST-1. In fetuses, BST-1+ cells appeared in the thymus and liver at day 14 and 16 of gestation respectively. The expression level of BST-1 by fetal thymus was maximal and > 60% of thymocytes were positive for BST-1 at day 15 or 16 and the proportion then gradually decreased during development. Among day 15 fetal thymocytes, BST-1 was negative on the CD44+ CD25- fraction, very slightly positive on the CD44+ CD25+ fraction, and strongly positive on the CD44(10)/- CD25+ and CD44-CD25- fractions. These results showed that murine BST-1 is a useful marker for lymphoid progenitor cells initiating gene rearrangement of their antigen receptors.

Medicinal foodstuff. III. Sugar beet. (1): Hypoglycemic oleanolic acid oligoglycosides, betavulgarosides I, II, III, and IV, from the root of Beta vulgaris L. (Chenopodiaceae).

Betavulgarosides I, II, III, and IV, oleanolic acid oligoglycosides having an unique acidic substituent, were isolated from the root of Beta vulgaris L. (sugar beet) together with betavulgarosides VI, VII, and VIII. The chemical structures of betavulgarosides I, II, III, IV were identified from chemical and physicochemical evidence. Betavulgarosides II, III, and IV were found to exhibit hypoglycemic activity in an oral glucose tolerance test in rats.

Elevated levels of the soluble form of bone marrow stromal cell antigen 1 in the sera of patients with severe rheumatoid arthritis.

OBJECTIVE: Bone marrow stromal cell antigen 1 (BST-1) is a novel glycosyl phosphatidylinositol-anchored ectoenzyme, which is overexpressed on bone marrow stromal and synovial cell lines derived from patients with rheumatoid arthritis (RA). To investigate the pathophysiologic roles of BST-1 in RA, we established an enzyme-linked immunosorbent assay (ELISA) system to detect the soluble form of BST-1 (sBST-1) and examined levels of sBST-1 in the sera of RA patients. METHODS: Concentrations of sBST-1 in sera from healthy donors and from patients with RA, osteoarthritis, Sjögren's syndrome, and systemic lupus erythematosus were measured with the ELISA. RESULTS: In 7% of the RA patient samples (10 of 143), concentrations of serum sBST-1 were higher (approximately 30-50-fold) than in non-RA samples. Serum sBST-1 concentrations showed no correlation with age, C-reactive protein level, or rheumatoid factor level. All RA patients with high concentrations of serum sBST-1 had severe disease with involvement of several large joints. CONCLUSION: We believe the measurement of serum sBST-1 may have prognostic value, but further analysis is necessary to clarify the clinical significance of elevated sBST-1 in RA.

Studies on antiulcer agents. III. Plausible mechanism of antisecretory action of ethyl 2[(1H-benzimadazol-2-yl)sulfinylmethyl]-4-dimethylamino- 5-pyrimidinecarboxylate, an H+/K(+)-ATPase inhibitor, based on its reaction with thiols.

To explore the mechanism of the gastric antisecretion activity of ethyl 2-[(1H-benzimidazol-2-yl)sulfinylmethyl]- 4-dimethylamino-5-pyrimidinecarboxylate (5), a potential H+/K(+)-ATPase inhibitor, in the acid compartment of parietal cells, its reaction with some alkylthiols in the presence of hydrochloric acid was investigated. Upon treatment with 2-mercaptoethanol under acidic conditions, 5 gave a characteristic 1:2 adduct, ethyl 4-[2-(2-hydroxyethyldithio)- 1-(2-hydroxyethylthio)ethylidenamino]pyrimido[1,2-a]benzimid azole-3- carboxylate (6), instead of providing a disulfide of type 3, 2-(2-alkyldithiomethylpyridino)benzimidazolide, the product predicted to be formed according to the reaction mechanism of common H+/K(+)-ATPase inhibitors, such as omeprazole or lansoprazole, with mercaptans. With a large excess of 2-mercaptoethanol, 5 provided 2-(2-hydroxyethylthio)-1H-benzimidazole (8) and ethyl 4-dimethylamino-2-(2-hydroxyethyldithio)-5-pyrimidinecarboxylat e (9) as well as 6. The transformation mechanisms and their implications are discussed.

Comparison of the in vitro skin penetration of propiverine with that of terodiline.

This study was designed to clarify the relationship between the properties of propiverine and skin penetration, and to compare the in vitro penetration characteristics of propiverine and terodiline through rat skin. Propiverine in both hydrochloride and free forms penetrated across the skin extremely slowly, with a 2.6 times higher flux in the hydrochloride than that in the free base, in the absence of enhancers. Various enhancers failed to enhance the penetration of propiverine hydrochloride, whereas the same agents slightly increased the flux of the free form, these being due to the slow release rate of the free form from the gel formulations, an extremely high lipophilicity (log Poct/water > 4.97), much less solubility (0.141 mg/ml) and a large partition capacity of the drug to skin components. Terodiline in both forms was able to rapidly penetrate through the skin, even in the absence of enhancers, with 20.2 and 9.8 times higher fluxes respectively, than the corresponding forms of propiverine. The high penetration characteristics of terodiline would be due to a suitable lipophilicity, low binding property as well as the structural masking from the binding to the epidermal components. Propiverine hydrochloride penetrated through the stratum corneum 4.4 times and viable skin 3.1 times higher than through full-thickness skin, while the fluxes of terodiline through the stratum corneum and viable skin were similar to each other, with high penetration rates for each form.