Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.     

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [(35)S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [(35)S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.     

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 +/- 0.7 degrees C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5 degrees C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 +/- 0.7 degrees C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.     

Synthesis of non-natural C2-homo-ceramide and its apoptotic activity against HL-60 cells

Non-natural ceramide analogues, C2-homo-ceramide and C2-homo-dihydroceramide, were prepared from L-aspartic acid via L-homo-serine. The apoptotic activities of the synthesized ceramide analogues were examined in HL-60 human leukemia cells. C2-homo- and C2-bishomo-ceramide indicate low but considerable apoptotic activities in comparison with C2-ceramide.     

Identification of a stable chymase inhibitor using a pharmacophore-Based database search

In general, serine protease chymase inhibitors readily decompose in plasma. We previously found that thiazolidine-2,4-dione and thiadiazole derivatives are also unstable. Using a pharmacophore-based database search, we identified a benzo[b]thiophen-2-sulfonamide derivative as a stable chymase inhibitor. Finding a lead compound with adequate activity and stability by a pharmacophore-based approach is more efficient than modifying an unstable compound to reduce its instability without simultaneously decreasing its inhibitory activity. Our pharmacophore model of chymase inhibitors suggests that the two hydrophobic interactions in the S1 and S1' regions and the two H-bonding interactions between them play important roles in chymase inhibitors.     

Apoptotic activities of C2-ceramide and C2-dihydroceramide homologues against HL-60 cells

The apoptotic activities of non-natural ceramide homologues, C2-homo-ceramide, C2-homo-dihydroceramide, C2-bishomo-ceramide and C2-bishomo-dihydroceramide, were examined using human leukemia HL-60 cells. The apoptotic activity was in order of C2-ceramide>C2-homo-ceramide approximately C2-bishomo-ceramide and the activities of the L-erythro- and D-erythro-ceramide homologues were similar. The morphological features of the cells, DNA fragmentations, proteolytic processing of pro-caspase-3 and the cleavage of PARP as the result of treatments with these homologues indicated that cell death was induced by apoptosis.