Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

The periodic loss of metabolically unstable DNA during replication of chromosomal DNA of CHO cells

The fate of ³H-thymidine incorporated into newly synthesized DNA of CHO cells was analyzed by either the estimation of the incorporated radioactivity per cell or sedimentation in alkaline sucrose gradient. Under conditions in which DNA synthesis proceeded continuously, of incorporated radioactivity was periodically lost and regained during a 90 min chase, corresponding to a cyclic change in the sedimentation profiles. When DNA synthesis was inhibited by hydroxyurea no cyclic change of the incorporated radioactivity was observed. The cyclic changes were regarded as the result of an actual metabolic change in³H-labelled DNA probaly joining to one of the newly formed sister strands of DNA and the loss of radioactivity seems to require active continued DNA synthesis.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method

Summary Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-pointdried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the threedimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0° C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0° C were further incubated at 37° C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cellbound ligands.This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, and the Japan Private School Promotion Foundation     

The biological activity of catfish pancreatic somatostatin

Catfish pancreatic somatostatin, which contains eight additional amino acids on the amino terminus of a tetradecapeptide with considerable homology to tetradecapeptide somatostatin (SRIF), is a naturally occurring homology of the hypothalamic peptide. The purpose of these studies was to determibe the biological activity of this somatostatin homolog. Inhibition of ¹²⁵I-labelled tyr¹-SRIF binding to bovine pituitart plasma membranes by catfish pancreatic somatostatin was approximately 33% that of SRIF. Pancreatic somatostatin has full biological activity measured by inhibition of growth hormone release from isolated rat pituitary cells, but 0.01–0.1% the potency of SRIF. Pancreatic somatostatin at 100 ng/ml produced a 50–60% inhibition of insulin and glucagon secretion from perfused rat pancreas, while SRIF produced comparable inhibition at 10 ng/ml. This report demonstrates that a larger molecular form and natural homolog of SRIF, isolated from fish pancreas, has the same (but reduced) biological activities in rat assay systems as somatostatin originally isolated from sheep hypothalamus.     

Effect of 6059-S,a novel oxacephem,on cross-linking reaction of peptidoglycan biosynthesis in Escherichia coli,Pseudomonas aeruginosa and Serratia marcescens

The effect of 6059-S, a novel 1-oxacephem, on peptidoglycan synthesis was investigated using ether-treated cells of Escherichia coli K 12, Pseudomonas aeruginosa KM 338 and Serratia marcescens IFO 12648. The cross-linking reaction of peptidoglycan synthesis in these organisms was inhibited by markedly low concentration of 6059-S.Non-standard abbreviations PBP penicillin binding protein - MIC minimum inhibitory concentration - ETB ether treated bacterial cells - SDS sodium dodecylsulfate