Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Induction of rapid testicular growth in Japanese quail by phasic electrical stimulation of the hypothalamic photosensitive area

Summary Optic fibers were implanted stereotaxically into the brain of immature male Japanese quail reared under short-day photoperiod (lights on from 1000 to 1800 h), and photosensitive sites in the hypothalamus were examined using gonadal growth and associated hormonal changes as the indices.In the subsequent experiments, bipolar (coaxial) electrodes were implanted chronically using predetermined coordinates for highly photosensitive sites. Henceforth the birds received brief electrical stimulation (square wave, 100 Hz, 100A, 2 min) once daily for 21 consecutive days. When the electrical stimulation was applied early in the dark period, marked gonadal growth was induced, but identical stimulation given in the light period resulted in no testicular growth. The response curve of testicular weight vs clock time of electrical stimulation has a prominent peak at 3 h after the onset of dark. Apparently, the neural complex in the photosensitive area of the quail hypothalamus responds to electrical stimulation as it does to light. We conclude that in photoperiodic birds the principal factor which determines the magnitude of gonadal responses is not the intensity of the stimulus but its timing (circadian phase).Abbreviations GnRH gonadotropin releasing hormone - LD light and dark - LED light emitting diode - CRT cathode ray tube - LH luteinizing hormone - OD outer diameter - ID inner diameter     

Molecular chaperones facilitate the soluble expression of <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolases in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone.     

Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction

Several studies have demonstrated that NF-kappaB is substantially involved in the progression of cardiac remodeling; however, it remains uncertain whether the continuous inhibition of NF-kappaB is effective for the prevention of myocardial remodeling. Myocardial infarction (MI) was produced by ligation of the left anterior coronary artery of rats. IMD-0354 (10 mg/kg per day), a novel phosphorylation inhibitor of IkappaB that acts via inhibition of IKK-beta, was injected intraperitoneally starting 24 h after induction of MI for 28 days. After 28 days, the IMD-0354-treated group showed significantly improved survival rate compared with that of the vehicle-treated group (P < 0.05). Although infarct size was similar in both groups, improved left ventricular (LV) remodeling and diastolic dysfunction, as indicated by smaller LV cavity (LV end-diastolic area: vehicle, 74.13 +/- 3.57 mm(2); IMD-0354, 55.00 +/- 3.73 mm(2); P < 0.05), smaller peak velocity of early-to-late filling wave (E/A) ratio (vehicle, 3.87 +/- 0.26; IMD-0354, 2.61 +/- 0.24; P < 0.05), and lower plasma brain natriuretic peptide level (vehicle, 167.63 +/- 14.87 pg/ml; IMD-0354, 110.75 +/- 6.41 pg/ml; P < 0.05), were observed in the IMD-0354-treated group. Moreover, fibrosis, accumulation of macrophages, and expression of several factors (transforming growth factor-beta1, monocyte chemoattractant protein-1, matrix metalloproteinase-9 and -2) in the noninfarcted myocardium was remarkably inhibited by IMD-0354. In conclusion, inhibition of NF-kappaB activation may reduce the proinflammatory reactions and modulate the extracellular matrix and provide an effective approach to prevent adverse cardiac remodeling after MI.     

Comparison between Holstein cow's milk and Japanese-Saanen goat's milk in fatty acid composition, lipid digestibility and protein profile

The fatty acid composition, the lipid digestibility and protein profile of Japanese-Saanen goat's milk were characterized. Caprine milk contained substantial quantities of C(4:0) to C(10:0) fatty acids as compared with Holstein cow's milk. The lipids of the former showed significantly higher digestibility in vitro by porcine lipase than those of the latter (P<0.05). As determined by SDS-PAGE, the respective contents of alpha(s1)-casein, one of the major allergens, were 3.9% and 33.7% in caprine and bovine milk.     

Discovery of 7alpha-substituted dihydrotestosterones as androgen receptor pure antagonists and their structure-activity relationships

A series of 7alpha-substituted dihydrotestosterone derivatives were synthesized and evaluated for androgen receptor (AR) pure antagonistic activity. From reporter gene assay (RGA), the compound with a side chain containing N-n-butyl-N-methyl amide (19a) showed pure antagonistic activity (IC(50)=340nM, FI(5)>10,000nM), whereas known AR antagonists showed partial agonistic activities. The optimization of 19a led to compound 23 (CH4892280), which showed more potent pure antagonistic activity (IC(50)=190nM, FI(5)>10,000nM). The SARs of tested compounds suggested that the length of the side chain and the substituents on the amide nitrogen are important for pure antagonistic activities.     

The mechanical binding strengths of Helicobacter pylori BabA and SabA adhesins using an adhesion binding assay–ELISA,and its clinical relevance in Japan

To elucidate a potential role for H. pylori BabA and SabA adhesins in the pathogenesis of gastric mucosal lesions, the MBS of BabA and SabA was examined using an in‐house ABA‐ELISA. Ninety isolates from Japanese patients with gastric cancer (n= 43) and non‐cancerous (n= 47) lesions were subjected to an ABA‐ELISA which had been developed in‐house, and sequential analysis of the babA2 middle region. The BabA‐MBS was significantly higher in the cancer than the non‐cancer group (P= 0.019), but there was no significant difference for SabA‐MBS. A weak correlation between BabA‐MBS and SabA‐MBS (r= 0.418) was observed, the positive correlation being higher in the cancer than the non‐cancer group (r= 0.598 and 0.288, respectively). The isolates were classified into two groups: a BabA‐high‐binding and a BabA‐low‐binding group (in comparison to the average for BabA‐MBS). The average SabA‐MBS in the BabA‐high‐binding group was significantly higher than in the BabA‐low‐binding group (P < 0.0001). Analysis of babA2 middle region diversity (AD1–5) revealed that AD2‐type was predominant in isolates irrespective of BabA‐MBS. H. pylori BabA‐MBS might have an effect on SabA‐MBS and relate to the severity of gastric disorders, including gastric cancer. Evaluation of MBS of the combined two adhesins would be helpful for predicting damage in the H. pylori infected stomach.     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.