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.

Antitumor agents. 44. Bis(helenalinyl) esters and related derivatives as novel potent antileukemic agents.

Bis(helenalinyl), bis(plenolinyl), bis(2,3-dihydrohelenalinyl), and bis(2,3,11,13-tetrahydrohelenalinyl) esters have been synthesized in an effort to elucidate the role of the two enone alkylating centers, beta-unsubstituted cyclopentenone and alpha-methylene gamma-lactone, as well as the significance of the diester linkage with respect to the enhanced in vivo P-388 lymphocytic leukemia antileukemic activity of bis(helenalinyl) malonate (2) against P-388 lymphocytic leukemia in the mouse. The bisesters (2-5; 7, 8; 10, 11) are, in general, more potent and less toxic than their corresponding parent alcohols (1, 6; 9; 14). The beta-unsubstituted cyclopentenone ring and the alpha-methylene gamma-lactone moiety in the bisesters play important roles for the enhancement of the P-388 antileukemic activity. Removal of the enone double bonds in both alkylating centers of 2 gave rise to inactive compounds. Except for 2, the potent antileukemic activity of the bis(helenalinyl) esters (3-5) appears to be independent of the ester chain length.

Transcriptional activation of the interleukin-6 gene by HTLV-1 p40tax through an NF-kappa B-like binding site.

The interleukin-6 (IL-6) gene is expressed by various stimuli including cytokines or viral infections, such as human T-cell leukemia virus type I (HTLV-1). However, it has not been well established how HTLV-1 induces the expression of the IL-6 gene. In the present study, we demonstrated that HTLV-1-derived transactivator protein, p40tax, could stimulate endogenous IL-6 gene expression. Furthermore, we showed that the NF-kappa B binding site (IL-6 kappa B site) located between -74 and -62 upstream of the cap site of the IL-6 gene was an essential cis-acting element for p40tax-mediated transactivation of the IL-6 gene expression by utilizing a series of 5' deletion mutants of the IL-6 5' flanking region as well as a construct with a mutated IL-6 kappa B site. We identified the presence of two nuclear factor complexes that bound to the IL-6 kappa B site. One was constitutively expressed, and the other was inducible by p40tax. Taken together, HTLV-1 p40tax directly induces IL-6 gene expression through the IL-6 kappa B site, indicating the close association between IL-6 overproduction and HTLV-1 infection.

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.

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.

Antihyperlipidemic activity of sesquiterpene lactones and related compounds.

Some naturally occurring pseudoguaianolides and germacranolides as well as synthetic related compounds were observed to be antihyperlipidemic agents in mice. Several of these compounds at a dose of 20 mg/kg/day resulted in lowering of serum cholesterol by approximately 30% and of serum triglycerides by approximately 25%. Thiol-bearing enzymes of lipid synthesis, i.e., acetyl-CoA, citrate-lyase, acetyl-CoA synthetase, and beta-hydroxy-beta-methylglutaryl-CoA reductase, were inhibited by these agents in vitro, supporting the premise that these agents alkylate thiol nucleophiles by a Michael-type addition. The alpha-methylene-gamma-lactone moiety, the beta-unsubstituted cyclopentenone ring, and the alpha-epoxycyclopentanone system of these compounds appeared to be responsible for the lowering of serum lipids.

Antitumor agents 68: effects of a series of helenalin derivatives on P-388 lymphocytic leukemia nucleic acid and protein synthesis.

A series of analogues related to helenalin demonstrated moderate capability for inhibiting the growth of murine P-388 lymphocytic leukemia cells in vivo and in vitro. The growth inhibition correlated with suppression of both DNA and protein synthesis in P-388 cells. The inhibition of protein synthesis occurred at a relatively low concentration and appeared to occur at the level of initiation. The suppression of DNA synthesis in P-388 cells correlated positively with inhibition of inosine 5'-monophosphate dehydrogenase activity. Although nuclear and alpha DNA polymerase activities were suppressed by certain analogues, the inhibition of the polymerases did not correlate positively with DNA synthesis inhibition and, furthermore, the magnitude of suppression of DNA polymerase activity did not appear to be sufficient to account for the observed suppression of DNA synthesis in P-388 cells.

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.

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.

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.

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.

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.

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.

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.

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.

Pharmacokinetic analysis of phenytoin and its derivatives in plasma and brain in rats.

The derivatives of phenytoin (DPH) were synthesized by the reaction at 3 position of hydantoin ring with valproic acid and valeric acid, producing valproyl DPH (VPDPH) and valeroyl DPH (VADPH), respectively. These derivatives showed much higher lipid solubilities than that of DPH. Their distribution and elimination were compared to those of DPH. Additionally, the concentration profiles of the drugs in brain and plasma were analyzed with a modified 2-compartment model. DPH and its derivatives, without hydrolysis to DPH in blood, were found rapidly distributed into brain, although the distribution of derivatives was much less, probably due to the high protein binding capacities. The distribution of DPH and its derivatives into brain regions was similar to that into the cortex cerebri. VPDPH and VADPH were more rapidly eliminated from plasma and brain than DPH, giving smaller mean residence time (MRT) values (0.92 and 0.85 h) and much smaller cortex/plasma concentration ratio than those of DPH. The VPDPH and VADPH concentrations in the cerebrospinal fluid (CSF) were also much lower than that of DPH. The time course of plasma and brain concentrations of DPH and its derivatives after i.v. administration was successfully described by the modified 2-compartment models presented.

Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth.

In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.

Physicochemical and hydrolytic characteristics of phenytoin derivatives.

To further clarify the pharmacokinetic characteristics of phenytoin (DPH) and its derivatives, DPH-1-methylnicotininate (MNDPH), valeroyl DPH (VADPH) and valproyl DPH (VPDPH), in plasma and brain, we have investigated their physicochemical properties and protein binding characteristics. Additionally, the hydrolytic conversion of these derivatives to DPH was also studied using small intestine, liver and brain tissues, as well as rat plasma. The log partition coefficient (PC) values of all derivatives were much higher than that of DPH. Judging from their pKa values (5.68 and 5.91 for VADPH and VPDPH, respectively) and pH-solubilities, VADPH and VPDPH were acidic compounds, while MNDPH was basic. These data indicated that most fractions of VADPH and VPDPH existed as an ionized form (these fractions existed in an ionized form, 0.98 and 0.97, respectively) at physiological pH, whereas MNDPH existed as a unionized form under the same conditions. Rosenthal or Scatchard plots of the binding data of DPH and its derivatives to both rat plasma protein and bovine serum albumin (BSA) exhibited straight lines over their concentration ranges used, indicating that DPH and its derivatives have a single binding site on the protein. The binding potencies (K or n.Pt value) of the derivatives to both proteins were much greater than that of DPH. No DPH produced from VADPH and VPDPH was found in the biological fluids over a period of 24 h. However, the hydrolysis of MNDPH to DPH was observed in plasma and the tissues used, with the most rapid hydrolysis in the small intestine, and the hydrolysis rate constant in plasma was ca. 20-fold greater than that in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

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.