Silver-catalyzed benzylation and allylation of tertiary alkyl bromides with organozinc reagents.

Silver salts catalyze the benzylation and allylation of tertiary alkyl bromides with organozinc reagents. The reactions create quaternary carbon centers efficiently. Treatment of gem-dibromoalkanes with benzylic or allylic zinc reagents under silver catalysis leads to dibenzylation or diallylation. The functional-group compatibility of the present reactions is wider than that of the previous reactions with Grignard reagents.

Residual motion and duty time in respiratory gating radiotherapy using individualized or population-based windows.

PURPOSE: The efficiency and precision of respiratory gated radiation therapy for tumors is affected by variations in respiration-induced tumor motion. We evaluated the use of individualized and population-based parameters for such treatment. METHODS AND MATERIALS: External respiratory signal records and images of respiration-induced tumor motion were obtained from 42 patients undergoing respiratory gated radiation therapy for liver tumors. Gating window widths were calculated for each patient, with 2, 4, and 10 mm of residual motion, and the mean was defined as the population-based window width. Residual motions based on population-based and predefined window widths were compared. Duty times based on whole treatment sessions, at various window levels, were calculated. The window level giving the longest duty time was defined as the individualized most efficient level (MEL). MELs were also calculated based on the first 10 breathing cycles. The duty times for population-based MELs (defined as mean MELs) and individualized MELs were compared. RESULTS: Tracks of respiration-induced tumor motion ranged from 3 to 50 mm. Half of the patients had larger actual residual motions than the assigned residual motions. Duty times were greater when based on individualized, rather than population-based, window widths. The MELs established during whole treatment sessions for 2 mm and 4 mm of residual motion gave significantly increased duty times, whereas those calculated using the first 10 breathing cycles showed only marginal increases. CONCLUSIONS: Using individualized window widths and levels provided more precise and efficient respiratory gated radiation therapy. However, methods for predicting individualized window levels before treatment remain to be explored.

Purification and characterization of a novel L-2-amino-Delta2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli.

L-2-Amino-Delta2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30-35 degrees C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the L-form of D,L-ATC and exhibited high activity for the hydrolysis of L-ATC with the values of Km (0.35 mM) and Vmax (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for D,L- and L-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon-sulfur bond in a ring structure of L-ATC to produce N-carbamoyl-L-cysteine.

Cloning and expression of 1,2-alpha-mannosidase gene (fmanIB) from filamentous fungus Aspergillus oryzae: in vivo visualization of the FmanIBp-GFP fusion protein.

1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.

A novel N-carbamoyl-L-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-L-cysteine amidohydrolase expressed in Escherichia coli.

N-carbamoyl-L-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50 degrees, respectively. The enzyme required Mn(2+) ion for activity expression and was inhibited by EDTA, Hg(2+) and sulfhydryl reagents. The enzyme was strictly specific for the L-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-L-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel L-carbamoylase, different from the known L-carbamoylases.

Identification, cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a.

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.

Induction of 2-amino-D2-thiazoline-4-carboxylic acid hydrolase and N-carbamoyl-l-cysteine amidohydrolase by S-compounds in Pseudomonas putida AJ3865.

The induction of 2-amino-Delta(2)-thiazoline-4-carboxylic acid hydrolase (ATCase) and N-carbamoylcysteine amidohydrolase (NCCase), both of which are involved in the conversion step of 2-amino-Delta(2)-thiazoline carboxylic acid (ATC) to cysteine, was studied with Pseudomonas putida AJ3865. We found that L-ATC induced L-ATCase and L-NCCase, but that D-ATC induced only L-NCCase, whereas L- or D-NCC and thiazoline derivatives did not induce both enzymes. The bacterium showed neither D-ATCase nor D-NCCase activities, indicating that the role of L-ATC and D-ATC was different in the enzyme induction. We also found new inducers, d- and l-methionine, S-methyl-L-cysteine, cysteic acid, and 2-aminoethane sulfonic acid. However, the induction level of both enzymes by new inducers was much lower than those by L-ATC and D-ATC. Furthermore, the induction rate of both enzymes was synergistically increased only under a combination of D,L-ATC and new inducers. S-Compounds, however, such as new inducers except S-methyl-L-cysteine, inhibited both enzyme activities. This is the first report on the new inducers, synergistic induction, and the new inhibitors of L-ATCase and L-NCCase.