Pharmacological studies on basic ethers (R111 and R97) with antispasmodic activity

Pharmacological properties and acute toxicity of 2-tolyl 1-phenyl-3-(2-methylpiperidino) propyl ether methyl bromide (R111) and 2-chlorophenyl 1-phenyl-3-(2-methylpiperidino) propyl ether methyl iodide (R97) were examined. The results obtained were as follows: (1) In the analgesic effects, RIII and R97 inhibited markedly the acetic acid-induced writhing in mice, but in reducing pain induced by heat, R111 and R97 showed negative results. The local anesthetic effect of R111 was approximately equal to that of procaine. R111 and R97 showed no effects on spontaneous locomotion, the convulsion induced by strychnine or pentetrazol, and normal body temperature. (2) R111 and R97 antagonized acetylcholine, barium chloride, nicotine and serotonine-induced spasm, but not that of histamine and bradykinin. In particular they possessed marked anti-barium chloride activity, where their effects were 20 to 30 times more active than that of papaverine. (3) R111 and R97 indicated weak mydriatic activity. (4) R111 and R97 showed inhibitory effects on the pilocarpine-induced sialic secretion and the propulsive movements of the small intestine, but their inhibitory effects on the gastric secretion were relatively weak. (5) R111 and R97 displayed protective effects in Shay's ulcer, but had no curative effects on acetic acid ulcer. (6) R111 and R97 induced temporary reduction of arterial blood pressure and blood flow immediately after the administration of the test compounds in anesthetized rabbits. However, these agents induced no change in ECG, heart rate and respiration. (7) Intraperitoneally administered R111 and R97 were effective in inhibiting the carrageenin-induced edema in the hind paw of rats. From the above results, it may be considered that R111 and R97 have together strong cholinergic blocking and muscotropic antispasmodic effects, moreover, no significant effects on the central nervous system.     

An application of Monte Carlo method to simulate disorders in polymer crystals

The Monte Carlo method is applied to polymer crystals of idealized linear chain molecules of 30 carbon atoms, and the unharmonic, large-amplitude, oscillations and the subsequent conformational disorders of the chains are investigated. A crystalline field that confines the chain is treated by the molecular field approximation, and assumed to be cylindrical in this work. A production type simulation is adopted taking into account rigorous statistical weights for each sample conformation. Both the rotational isomeric model and the continuous rotation model of chain conformation are considered. By averaging over 10⁴–8 × 10⁴ chains, mean-square end-to-end distance, fractions of gauche and trans states and a detailed distribution of internal rotation angle are obtained. The effects of temperature and pressure on the conformation of the chain in the crystals are also simulated.     

Severe ethanol stress induces assembly of stress granules in Saccharomyces cerevisiae

Stress granules (SGs) and processing bodies (P bodies) are cytoplasmic domains and play a role in the control of translation and mRNA turnover in mammalian cells subjected to environmental stress. Recent studies have revealed that SGs also form in the budding yeast Saccharomyces cerevisiae in response to glucose depletion and robust heat shock. However, information about the types of stress that cause budding yeast SGs is quite limited. Here we demonstrate that severe ethanol stress generates budding yeast SGs in a manner independent of the phosphorylation of eIF2α. The concentration that generated budding yeast SGs (>10%) was higher than that causing P bodies (>6%), and P bodies were assembled prior to SGs. As well as mammalian SGs, the assembly of budding yeast SGs under ethanol stress was blocked by cycloheximide. On the other hand, the budding yeast SGs caused by ethanol stress contained eIF3c but not eIF3a and eIF3b, although the eIF3 complex is a core constituent of mammalian SGs. Moreover, null mutants (pbp1Δ, pub1Δ and tif4632Δ) with a strong reduction in SG formation did not resume proliferation after the elimination of ethanol stress, indicating that the formation of budding yeast SGs might play a role in sufficient recovery from ethanol stress.     

An outbreak of food-borne listeriosis due to cheese in Japan, during 2001

Food-borne outbreaks caused by Listeria monocytogenes have been recognized in US and European countries. Only sporadic cases, of neonatal listeriosis, have been reported in Japan. Since L. monocytogenes has been often isolated from foods in Japan, food-borne outbreaks potentially could have occurred. In February 2001, L. monocytogenes serotype 1/2b was isolated from a washed-type cheese during routine Listeria monitoring of 123 domestic cheeses. Further samples from products and the environments at the plant that produced the contaminated cheese were examined for L. monocytogenes. L. monocytogenes serotype 1/2b was detected in 15 cheese samples, at most probable number that ranged from <30 to 4.6 x 10(9)/100 g, and in environmental samples. Studies with people who had consumed cheese from the plant revealed 86 persons who had been infected with L. monocytogenes. Thirty-eight of those people had developed clinical symptoms of gastroenteritis or the common cold type after the consumption of cheese. Isolates from those patients exhibited the same serotype, pathogenicity for mice and HeLa cells, DNA fingerprinting patterns and PCR amplification patterns. From the epidemiological and genetic evidence, it appeared that the outbreak was caused by cheese. This is the first documented incidence of food-borne listeriosis in Japan.     

Antimicrobial activity of nutmeg against Escherichia coli O157

We examined the difference between Escherichia coli O157 and non-pathogenic E. coli in their tolerance to spices. Various spices (5 g each) were homogenized at 25 degrees C for 10 min with 5 ml of 70% ethyl alcohol, and the supernatant solutions obtained by centrifugation were used as spice extracts. When the E. coli strains were incubated with each spice extract at concentrations of 0.01% and 0.1%, a noteworthy difference was observed between the O157 and non-pathogenic strains in their tolerance to nutmeg. The populations of the non-pathogenic strains could not be reduced, but those of the O157 strains were remarkably reduced. Antibacterial activity by the nutmeg extract was also found against the enteropathogenic E. coli O111, but not against enterotoxigenic (O6 and O148) and enteroinvasive (O29 and O124) E. coli. When we examined the antibacterial effect of volatile oils in nutmeg on the O157 and non-pathogenic E. coli strains, all O157 strains tested were found to be more sensitive to beta-pinene than non-pathogenic E. coli strains.     

Steroid hormone profiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionization and atmospheric pressure photoionization sources

A highly sensitive and uncomplicated method of analyzing steroidal hormones in river and estuarine water samples was developed using a liquid chromatography tandem mass spectrometer equipped with an electrospray ionization (ESI) source and atmospheric pressure photoionization (APPI) source. Steroidal hormones included not only estrogen but also androgen and conjugates of these two. APPI displayed greater sensitivity than ESI for most of the unconjugated steroids examined, with very high sensitivity for testosterone and 4-androstene-3,17-dione in particular. For conjugated hormones, in contrast, ESI was more effective. The method developed was applied to the determination of hormones in the rivers of Osaka City and their estuaries, where the hormones detected were affected by the effluent from municipal wastewater treatment plants (WWTPs), and hormone concentration values were comparable to those reported in previous studies of such effluent. Because of the two-way flow and stagnancy of streams and watercourses, continuous input of steroidal hormones from WWTPs seems to bring about local accumulation. Levels of androgen were 1 order of magnitude lower than those of estrogen. Estrone, estrone 3-sulfate, and 4-androstene-3,17-dione were detected in almost all water samples, with maxima of 51, 5.1, and 6.4 ng L(-1), respectively.     

Novel poly-gamma-glutamate-processing enzyme catalyzing gamma-glutamyl DD-amidohydrolysis

The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endo-peptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1,000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.     

Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3

Structural-based mutational analysis of salt-tolerant glutaminase from Micrococcus luteus K-3 (Micrococcus glutaminase) revealed that three amino acid residues, S64, K67, and E160, were essential to a catalytic reaction. The result suggested that Micrococcus glutaminase had a possible catalytic mechanism similar to class A beta-lactamase rather than glutaminase-asparaginase from Pseudomonas 7A.     

Anthocyanins,but not Anthocyanidins,from Bilberry (Vaccinium myrtillus L.) Alleviate Pruritus via Inhibition of Mast Cell Degranulation

Abstract: We have previously reported that bilberry anthocyanins exhibit an anti‐pruritic effect in a mouse model of allergic contact dermatitis. It has been reported that anthocyanins are particularly sensitive to thermal treatment and are easily hydrolyzed to anthocyanidins when exposed to high temperatures. The objective of this study was to compare the anti‐pruritic effect of anthocyanin‐rich quality‐controlled bilberry extract and anthocyanidin‐rich degraded extract using a mouse model of allergic contact dermatitis. BALB/c mice with allergic contact dermatitis induced by 4 weeks of repeated application of 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) were administered Bilberon‐25 orally for 4 weeks after sensitization with TNCB. The effect of Bilberon‐25 on pruritus was evaluated by measurement of scratching behavior. RBL‐2H3 mast cells were used to investigate the effect of Bilberon‐25 on degranulation in 48/80‐stimulated mast cells. Compared with nonheated Bilberon‐25, the proportion of anthocyanins in heated Bilberon‐25 decreased, and the proportion of anthocyanidins was increased in heated‐time dependent manner. Treatment with non‐heated Bilberon‐25 significantly attenuated the TNCB‐induced increase in scratching behavior, whereas treatment with 2 h‐heated Bilberon‐25 did not. Moreover, 300 μg/mL nonheated Bilberon‐25 showed significant inhibition of degranulation in RBL‐2H3 mast cells, whereas 2 h‐heated Bilberon‐25 had no effect at any concentration studied. It is assumed that the inhibitory effect of bilberry anthocyanins on pruritus might be mediated, at least in part, by its inhibitory effect on mast cell degranulation. In conclusion, the anthocyanin‐rich but not anthocyanidin‐rich bilberry extract may be a useful dietary supplement for skin diseases involving pruritic symptoms, such as chronic allergic contact dermatitis, atopic dermatitis, and rhinitis.