Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Chlorination of cellulose with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in lithium chloride-N,N-dimethylacetamide.

Microcrystalline cellulose was chlorinated with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in LiCl-N,N-dimethylacetamide. At the early stage of the reaction only replacement of the 6-hydroxyl groups with chlorine was observed, and 3-hydroxyl groups were replaced at a lower rate with Walden inversion. The effects of reaction conditions on the extent of chlorination were studied in detail. More than two equivalents of chlorination reagents per glucose residue were necessary to attain a high degree of substitution (ds) by chlorine, and the maximum ds attained was 1.86. Chlorinated disaccharides were found in the hydrolyzates of chlorodeoxycelluloses hydrolyzed under mild conditions, and their structures were studied by mass spectrometry.     

Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones, from Fusarium oxysporum.

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of aldonate lactones to the corresponding aldonic acids, was purified 10-fold to apparent homogeneity, with a 61% overall recovery, from Fusarium oxysporum AKU 3702, through a purification procedure comprising DEAE-Sephacel, octyl-Sepharose CL-4B and hydroxyapatite chromatographies and crystallization. The molecular mass of the native enzyme, as estimated by high-performance gel-permeation chromatography, is 125 kDa, and the subunit molecular mass is 60 kDa. The enzyme contains 15.4% (by mass) glucose equivalent of carbohydrate, and about 1 mol calcium/subunit. The enzyme hydrolyzes aldonate lactones, such as D-galactono-gamma-lactone and L-mannono-gamma-lactone, stereospecifically. Furthermore, it can catalyze the asymmetric hydrolysis of D-pantoyl lactone, which is a promising chiral building block for the chemical synthesis of D-pantothenate. These reactions are reversible, and the reaction equilibrium at pH 6.0 has a molar ratio of nearly 1:1 with D-pantoyl lactone and D-pantoic acid. The Km and Vmax for D-galactono-gamma-lactone are 3.6 mM and 1440 U/mg, respectively, and those for D-galactonate are 52.6 mM and 216 U/mg, respectively. The enzyme also irreversibly hydrolyzes several aromatic lactones, such as dihydrocoumarin and homogentisic-acid lactone.     

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Suppression of the development of uterine adenomyosis by danazol treatment in mice.

Inhibitory effects of danazol, an isoxazol derivative of synthetic steroid 17 alpha-ethinyl-testosterone, on the development of uterine adenomyosis, a pathological disorder of endometrial tissue defined as the presence of endometrial glands and stroma in the myometrium, were investigated in mice of SHN strain. Mice treated with 0.5 microgram danazol for 5 weeks during 4-9 weeks of age and killed at 21 weeks of age showed significantly lower incidence of the spontaneous development of adenomyosis than the age-matched intact control mice. The inhibitory effects of danazol were also evident in mice bearing pituitary isografts which were effective in inducing an early and a high incidence of adenomyosis. Furthermore, the treatment with danazol resulted in the decrease of serum levels of luteinizing hormone (LH) and prolactin (PRL) associated with hypofunction of ovaries and persistent diestrus. These results support the usefulness of danazol for the clinical treatment of gynecological disorders except for hypofunction of ovaries.     

Explant organ culture: A review

Organ explant culture models offer several significant advantages for studies of patho-physiologic mechanisms like cell injury, secretion, differentiation and structure development. Organs or small explants/slices can be removed in vivo and maintained in vitro for extended periods of time if careful attention is paid to the media composition, substrate selection, and atmosphere. In the case of human tissues obtained from autopsy or surgery, additional attention must be paid to the postmortem interval, temperature, hydration, and cause of death. Explant organ culture has been effectively utilized to establish outgrowth cell cultures and characterize the histiotypic relationships between the various cell types within an organ or tissue.J. Resau is a visiting scientist at the NCI-LMO-DCE in Frederick, MD 21702, U.S.A.K. Sakamoto is a visiting scientist from the Department of Surgery, Gunma University School of Medicine, Maebashi, Japan     

Organomercurial-volatilizing bacteria in the mercury-polluted sediment of Minamata Bay, Japan.

A total of 4,604 bacterial strains isolated from the sediments of Minamata Bay and nearby low-level-mercury stations (control stations) were screened for the ability to volatilize mercury from inorganic and organic mercurial compounds. The strains that volatilize mercury from several kinds of organomercurials were found only in the sediments of Minamata Bay.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.