Structural Analysis of Sydowic Acid by X-ray Diffraction

Streptomyces sp. No. 280 produced several kinds of amylase inhibitors (amylase inhibitor A, B, B' and C). Two amylase inhibitors (designated as AI-A₁ and AI-A₂) were obtained from an amylase inhibitor A fraction by paper chromatography. AI-A₁ inhibited muscle phosphorylase a much more than AI-A₂ and was hydrolyzed by sweet potato β-amylase whereas AI-A₂ was not. Both amylase inhibitors had a carbohydrate and were hydrolyzed by some kinds of amylases or acids. They lost their inhibitory activity against phosphorylase a after treatment with acids or hog pancreatic α-amylase, but they showed increased inhibitory activity toward porcine small intestinal sucrase.

Both AI-A₁ and AI-A₂ were composed of glucose and a basic moiety which gave a positive ninhydrin reaction. The molecular weights of AI-A₁ and AI-A₂ were estimated to be approximately 1300 ? 1500 by gel filtration on a Sephadex G-15 column. The nitrogen content of the amylase inhibitors was found to be about 1.3% by elementary analysis     

Free radical scavenging activity of propolis

We investigated the radical scavenging activity of propolis by ESR spectroscopy using spin trapping method. In addition, we examined the influence of a diet of 2% propolis on mice under oxidative stress. At low concentrations, the methanolic extract of propolis exhibited strong scavenging activity in vitro towards both the superoxide anion radical, generated by the hypoxanthine-xanthine oxidase reaction, and the NO radical, generated from the mixture of NOC-7 (NO generator) and carboxy-PTIO (spin trapping agent). An inhibitory effect of propolis on lipid peroxidation in vivo was observed, as determined by measurement of thiobarbituric acid-reactive substances in mouse liver homogenate. The level of vitamin C in the brain of mice under oxidative stress significantly increased compared with control mice under atmosphere, which was not observed in the mice given 2% propolis. The level of alpha-tocopherol in the brain of mice given 2% propolis significantly increased compared with control mice under atmosphere, which was not observed in mice under oxidative stress. SOD activity in the brain and plasma of mice given 2% propolis significantly decreased under atmosphere and oxidative stress compared with control mice. These results suggest that propolis possesses potent antioxidant activity in vitro and in vivo.     

Neurotoxic mechanisms triggered by Alzheimer's disease-linked mutant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin target

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Identification of the mouse H-ficolin gene as a pseudogene and orthology between mouse ficolins A/B and human L-/M-ficolins

Ficolin is a collagenous lectin which plays a crucial role in innate immunity. Three and two ficolins have been identified in human and mice, respectively. To identify the mouse homologue of human H-ficolin and to elucidate the orthology between mouse ficolins A/B and human L-/M-ficolins, the gene structures were explored. The mouse homologue of the H-ficolin gene was identified as a pseudogene on chromosome 4. The mouse ficolin A gene was located far from the ficolin B gene on chromosome 2, whereas the human L-ficolin and M-ficolin genes were close in the region homologous to the ficolin B locus. Together with the exon-intron structures and the phylogenetic tree, these results suggest that ficolin B is the mouse orthologue of M-ficolin and that the genes encoding serum-type ficolins, ficolin A and L-ficolin, were generated independently from the ficolin B/M-ficolin lineage each in mice and primates.     

Enzyme immunoassay for serum 18-hydroxycorticosterone and its clinical application

An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3-(O-carboxymethyl)oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n=6) and 5.8% (n=6), respectively. Four of 5 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels.     

Pathogenesis of Hepatitis C Virus Infection in Tupaia belangeri

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.Hepatitis C virus (HCV) is a small enveloped virus that causes chronic hepatitis worldwide (32). HCV belongs to the genus Hepacivirus of the family Flaviviridae. Its genome comprises 9.6 kb of single-stranded RNA of positive polarity flanked by highly conserved untranslated regions at both the 5′ and 3′ ends (4, 27, 29). The 5′ untranslated region harbors an internal ribosomal entry site (29) that initiates translation of a single open reading frame encoding a large polyprotein comprising about 3,010 amino acids (35). The encoded polyprotein is co- and posttranslationally processed into 10 individual viral proteins (15).In most cases of human infection, HCV is highly potent and establishes lifelong persistent infection, which progressively leads to chronic hepatitis, liver steatosis, cirrhosis, and hepatocellular carcinoma (9, 16, 21). The most effective therapy for treatment of HCV infection is administration of pegylated interferon combined with ribavirin. However, the combination therapy is an arduous regimen for patients; furthermore, HCV genotype 1b does not respond efficiently (19). The prevailing scientific opinion is that a more viable option than interferon treatment is needed.The chimpanzee is the only validated animal model for in vivo studies of HCV infection, and it is capable of reproducing most aspects of human infection (5, 18, 23, 28, 35, 36). The chimpanzee is also the only validated animal for testing the authenticity and infectivity of cloned viral sequences (8, 14, 35, 36). However, chimpanzees are relatively rare and expensive experimental subjects. Cross-species transmission from infected chimpanzees to other nonhuman primates has been tested but has proven unsuccessful for all species evaluated (1).The tupaia (Tupaia belangeri), a tree shrew, is a small nonprimate mammal indigenous to certain areas of Southeast Asia (6). It is susceptible to infection with a wide range of human-pathogenic viruses, including hepatitis B viruses (13, 20, 31), and appears to be permissive for HCV infection (33, 34). In an initial report, approximately one-third of inoculated animals exhibited acute, transient infection, although none developed the high-titer sustained viremia characteristic of infection in humans and chimpanzees (33). The short duration of follow-up precluded any observation of liver pathology. In addition to the putative in vivo model, cultured primary hepatocytes from tupaias can be infected with HCV, leading to de novo synthesis of HCV RNA (37). These reports strongly support tupaias as a valid model for experimental studies of HCV infection. However, longitudinal analyses evaluating the clinical development and pathology of HCV-infected tupaias have yet to be examined. In the present study, we describe the clinical development and pathology of HCV-infected tupaias over an approximately 3-year time course.     

Elevation of histidine decarboxylase activity in skeletal muscles and stomach in mice by stress and exercise

The effects of different types of stress (water bathing, cold, restraint, and prolonged walking) on histidine decarboxylase (HDC) activity in masseter, quadriceps femoris, and pectoralis superficial muscles, and in the stomach were examined in mice. All of these stresses elevated gastric HDC activity. Although water bathing, in which muscle activity was slight, was sufficiently stressful to produce gastric hemorrhage and to increase gastric HDC activity, it produced no detectable elevation of HDC activity in any of the muscles examined. The other stresses all elevated HDC activity in all three muscles. We devised two methods of restraint, one accompanied by mastication and the other not. The former elevated HDC activity in the masseter muscle, but the latter did not. These results suggest that 1) HDC activity in the stomach is an index of responses to stress, 2) the elevation of HDC activity in skeletal muscles during stress is induced partly or wholly by muscle activity and/or muscle tension, and 3) stress itself does not always induce an elevation of HDC activity in skeletal muscles.