Acid-Catalyzed nucleophilic substitution and racemization of 3-methoxy-N-desmethyldiazepam enantiomers in methanol

pK_a1 values of 3-methoxy-N-desmethyldiazepam in acetonitrile and methanol containing various acid concentrations were determined by spectrophotometry to be 3.5 and 1.3, respectively. Temperature-dependent racemization of enantiomeric 3-methoxy-N-desmethyldiazepam in methanol containing 0.5 M H₂SO₄ was studied by circular dichroism spectropolorimetry and the racemization reactions were found to follow apparent first-order kinetics. Thermodynamic parameters of the racemization reaction were found to be: E_act = 18.8 kcal/mol, and at 25°C: ΔH^? = 18.3 kcal/mol, ΔS^? = ?14.8 entropy unit, and ΔG^? = 22.7 kcal/mol, respectively. The racemization had an isotope effect (k_H/k_D) of 1.6 at 42°C. Based on the results of this report and those of earlier reports by other investigators, a nucleophilically solvated C3 carbocation intermediate resulting from either a P (plus) or an M (minus) conformation is proposed to be an intermediate and responsible for the stereoselective nucleophilic substitution and the subsequent racemization of 3-methoxy-N-desmethyldiazepam enantiomers. © 1993 Wiley-Liss, Inc.     

Sub-Cellular Distribution of Zinc in Different Vegetative Organs and Their Contribution to Grains Zinc Accumulation in Rice Under Different Nitrogen and Zinc Supply

Journal of Plant Growth Regulation - Zinc is an important micronutrient for the growth and development of human body and plants. Proper use of nitrogen fertilizer and foliar application of Zn have...     

Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.

The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.     

一种具有纤溶活性的枯草杆菌蛋白激酶的研究──Ⅰ高产酶菌株的筛选与鉴定

本文主要阐述了一种具有纤溶活性的枯草杆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）蛋白激酶产生菌株的筛选与鉴定的研究结果。作者从初筛的１２株Ｂａｃｉｌｌｕｓｓｕｂｌｉｌｉｓ菌中,通过对固体发酵和液体发酵所产生的枯草杆菌蛋白激酶,用琼脂糖－纤维蛋白平板法测其活性,经比较不同菌株的活性,筛选出两株高产酶菌株：Ｂ．ｓｕｂｔｉｌｉｓＨＷ—１２和Ｂ．ｓｕｂｔｉｌｉｓＨＷ—３。同时对菌体和菌落形态特点、生理生化反应进行了鉴定,认为Ｂ．ＳｕｂｔｉｌｉｓＨＷ－１２菌株可用来做为发酵生产该酶的菌种。     

Isolation,characterization and application of a species-specific repeated sequence from Haynaldia villosa

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.     

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.     

Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products

Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third α-helix and three adjacent amino acids in the third repeat (R3) of c-Myb were replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding.