1H-Imidazo[4,5-c]quinoline derivatives as novel potent TNF-alpha suppressors: synthesis and structure-activity relationship of 1-, 2-and 4-substituted 1H-imidazo[4,5-c]quinolines or 1H-imidazo[4,5-c]pyridines

Structural modification of imiquimod (1), which is known as an interferon-alpha (IFN-alpha) inducer, for the aim of finding a novel and small-molecule tumor necrosis factor-alpha (TNF-alpha) suppressor and structure-activity relationship (SAR) are described. Structural modification of a imiquimod analogue, 4-amino-1-[2-(1-benzyl-4-piperidyl)ethyl-1H-imidazo[4,5-c]quinoline (2), which had moderate TNF-alpha suppressing activity without IFN-alpha inducing activity, led to a finding of 4-chloro-2-phenyl-1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]quinoline (10) with potent TNF-alpha suppressing activity. The relation between conformational direction of 2-(4-piperidyl)ethyl group at position 1 and TNF-alpha suppressing activity is also demonstrated by NMR.     

Gliclazide protects pancreatic beta-cells from damage by hydrogen peroxide

Oxidative stress is induced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. Recently, it has become aware that susceptibility of pancreatic beta-cells to oxidative stress contributes to the progressive deterioration of beta-cell function in type 2 diabetes. A hypoglycemic sulfonylurea, gliclazide, is known to be a general free radical scavenger and its beneficial effects on diabetic complications have been documented. In the present study, we investigated whether gliclazide could protect pancreatic beta-cells from oxidative damage. One hundred and fifty microM hydrogen peroxide reduced viability of mouse MIN6 beta-cells to 29.3%. Addition of 2 microM gliclazide protected MIN6 cells from the cell death induced by H(2)O(2) to 55.9%. Glibenclamide, another widely used sulfonylurea, had no significant effects even at 10 microM. Nuclear chromatin staining analysis revealed that the preserved viability by gliclazide was due to inhibition of apoptosis. Hydrogen peroxide-induced expression of an anti-oxidative gene heme oxygenase-1 and stress genes A20 and p21(CIP1/WAF1), whose induction was suppressed by gliclazide. These results suggest that gliclazide reduces oxidative stress of beta-cells by H(2)O(2) probably due to its radical scavenging activity. Gliclazide may be effective in preventing beta-cells from the toxic action of reactive oxygen species in diabetes.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.     

Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.