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We report two methods for the preparation of peptide thioesters containing Tyr(SO 3 H) residue(s), without use of a protecting group for the sulfate moiety. The first was based on direct thioesterification using carbodiimide on a fully protected peptide acid, prepared on a 2-chlorotrityl (Clt) resin with fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc-SPPS). Subsequent deprotection of the protecting groups with trifluoroacetic acid (TFA) (0 °C, 4 h) yielded peptide thioesters containing Tyr(SO 3 H) residue(s). Peptide thioesters containing one to three Tyr(SO 3 H) residue(s), prepared by this method, were used as building blocks for the synthesis of the N α -Fmoc-protected N-terminal part of P-selectin glycoprotein ligand 1 (PSGL-1) (Fmoc-PSGL-1(43-74)) via silver-ion mediated thioester segment condensation. The other method was based on the thioesterification of peptide azide, derived from a peptide hydrazide prepared on a NH 2 NH-Clt-resin with Fmoc-SPPS. Peptide thioester containing two Tyr(SO 3 H) residues, prepared via this alternative method, was used as a building block for the one-pot synthesis of the N-terminal extracellular portion of CC-chemokine receptor 5 (CCR5(9-26)) by native chemical ligation (NCL). The two methods for the preparation of peptide thioesters containing Tyr(SO 3 H) residue(s) described herein are applicable to the synthesis of various types of sulfopeptides.

The name "Actinomyces suis" was applied to each actinomycete isolate from swine actinomycosis by Grässer in 1962 and Franke in 1973. Nevertheless, this specific species was not included in the "Approved List of Bacterial Name" due to absence of the type cultures. Therefore, "Actinomyces suis" based on the description of Franke 1973 has been considered as "species incertae sedis". We isolated a number of Actinomyces strains from swine. The representative strains of them was designated as Chiba 101 that was closely similar to the description in "Actinomyces suis" reported by Franke in 1973. Interestingly, it was found that the biological characteristics of these strains were also very similar to those of Actinomyces denticolens. Furthermore, the average nucleotide identity (ANI) value between strain Chiba 101 and the type-strain of Actinomyces denticolens (=DSM 20671 T ) was found to be 99.95%. Sequences of the housekeeping genes and 16S rRNA gene showed 100% homology. These results strongly suggested that "Actinomyces suis" Franke 1973 is the same species as Actinomyces denticolens. Since actinomycosis caused by Actinomyces denticolens have been demonstrated in horses recently, it is necessary to recognize that Actinomyces denticolens is the pathogenic actinomycetes in broader range of animals.

Actinomyces sp. strain Chiba101, isolated from an arthritic leg joint of a pig raised in Japan, is a bacterium closely related to Actinomyces denticolens Here, we deciphered the complete genome sequence of Actinomyces sp. Chiba101 and the high-quality draft genome sequence of A. denticolens DSM 20671 T ., (Copyright © 2017 Kanesaki et al.)

To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450 lanosterol 14 alpha-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL-6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein showed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14 alpha-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14-alpha-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital- and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals.

Regulation of the activity of lanosterol 14 alpha-demethylase (cytochrome P-45014DM) by dietary cholesterol was studied in rats. In male rats fed a 3% cholesterol diet for 1 and 4 weeks, the activity of 24,25-dihydrolanosterol 14 alpha-demethylase was decreased in the liver. The cytochrome P-45014DM protein content detected by immunoblotting was also decreased by cholesterol feeding. These results demonstrate that dietary cholesterol acts as a repressive factor for lanosterol 14 alpha-demethylase. Further, the activity was enhanced by preincubation with phosphatase of the enzyme solution from both the control and cholesterol fed rats at the same rate. This result suggests that regulation of the activity involves phosphorylation of this enzyme.

Cytochrome P-450(14DM) catalyzing lanosterol 14 alpha-demethylation was purified from rat liver and was characterized by its catalytic properties and mode of expression. The purified protein, with an apparent molecular mass of 51 kDa, showed 24,25-dihydrolanosterol 14 alpha-demethylation activity (3.05 nmol/min/mg protein) in a reconstituted system with an apparent Km value of 40 microM. The reconstituted enzyme converted 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol (the oxygenated intermediates of 24,25-dihydrolanosterol) to 4,4-dimethylcholesta-8,14-dien-3 beta-ol (the 32-nor compound). Immunoblot analysis of liver microsomes from male and female rats showed that the enzyme protein was expressed at an early stage of development and attained its maximum at 4 weeks after birth, retaining the same level thereafter. No difference was evident between males and females in the level of cytochrome P-450(14DM) in the liver up to 7 weeks after birth.

Metabolism of 32-oxo-24,25-dihydrolanosterols (3 beta-hydroxylanost-8-en-32- al (4,delta 8-CHO) and 3 beta-hydroxylanost-7-en-32-al (5,delta 7-CHO)) was studied in a reconstituted system consisting of rat liver partially purified cytochrome P-450, which catalyzes lanosterol 14-demethylation (P-450(14DM)), and reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase. The reconstituted system converted delta 8-CHO to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol (2, 8, 14-Diene), which corresponds to the 14-deformylated product. delta 7-CHO, the isomer of delta 8-CHO, was not converted to the corresponding 14-deformylated product. The apparent Km value of cytochrome P-450(14DM) for delta 8-CHO was about 1/20 of that for 24,25-dihydrolanosterol (1, DHL). The metabolism of delta 8-CHO was inhibited by 7-oxo-24,25-dihydrolanosterol (6, 7-oxo-DHL), which is a potent inhibitor of cholesterol biosynthesis from lanosterol or DHL. However, the metabolism of delta 8-CHO was less inhibited by 7-oxo-DHL than that of DHL.

27-Nor-24,25-dihydrolanosterol (27-nor-DHL), 26,27-dinor-24,25- dihydrolanosterol (26,27-dinor-DHL), and 25,26,27-trinor-24,25-dihydrolanosterol (25,26,27-trinor-DHL), analogs of 24,25-dihydrolanosterol (DHL) which have no C-27 carbon, C-26,27 carbons and C-25,26,27 carbons, were converted to the corresponding 14-demethylated products using a reconstituted monooxygenase system from rat liver microsomes which contained cytochrome P-450(14DM) catalyzing lanosterol 14-demethylation and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase in the presence of NADPH and molecular oxygen. Each metabolite showed a relative retention time (RtR) of 0.72 with respect to each substrate in high-performance liquid chromatography (HPLC) on a reversed-phase column. Comparison of each gas chromatography-mass spectrum and RtR value with those of the metabolite of DHL, 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, indicated that the metabolites could be inferred to be 27-nor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, 26,27-dinor-4,4- dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, and 25,26,27-trinor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol. However, 24,25,26,27-tetranor- and 23,24,25,26,27-pentanor analogs of DHL and 20-iso-24,25-dihydrolanosterol were not metabolized by the reconstituted enzyme system.