Analysis of N-acetoxy-N-2-acetylaminofluorene-induced frame-shifts in pBR322

We present a simple system which can be used to study directly directly the sequence change and the cellular repair functions involved in frame-shift mutagenesis by a covalently reactive mutagen. Positive (+S) and negative (?S) alterations in the number of base pairs of the Tc gene of pBR322 were generated and particular clones with Ap^RTc^S phenotypes were selected for mutagenesis experiments. Exposure of these frame-shifted plasmid DNAs to a potent carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), in vitro, caused covalent alterations to DNA sequence and resulted in a number of revertants (Ap^RTc^R) not observed in the untreated controls. The dose curve indicated an exponential response suggesting single-hit kinetics. Differential inactivation of the Ap gene was observed among various E. coli strains. The wild-type AB1157 and AB2463 (yrecA) showed a similar dose curve while AB1886 (uvrA) showed a marked decrease in Ap^R clones at the same dose. Both addition (+S) and deletion (?S) plasmids exhibited similar dose curves on inactivation of Ap gene. The reversion frequency, however, of ?S plasmid was a factor of 10 times higher than +S plasmid. The reversion frequency also increase markedly with uvrA host but not with recA host. 2 types of deletion revertants of the +S plasmid were found. 1 revertant has a single GC base-pair deletion in GC-rich region which is likely to be a target for AAAF reaction. The other showed a deletion of 4 base pairs (TCGA) at the tandem repeating sequence TCGATCGA which may represent a hot spot for frame-shift mutation.     

Sciatin Is a Transferrin-Like Polypeptide

Abstract: Sciatin, an acidic glycoprotein from chicken sciatic nerve, has myotrophic effects on avian skeletal muscle cells in culture. As sciatin was found to have certain structural similarities to transferrin, we further investigated the physicochemical characteristics of sciatin in order to determine the relationship between these two proteins. Sciatin was found to be strikingly similar to ovotransferrin in amino acid composition. In addition, amino acid sequence analysis revealed that sciatin and ovotransferrin had identical amino-terminal sequences for at least the first 20 amino acid residues. Chicken ovotransferrin, but not human serum transferrin, cross-reacted with rabbit antisciatin antibodies upon rocket immunoelectrophoresis and double immunodiffusion in agar. In addition, in the presence of bicarbonate, sciatin bound approximately 2 mol ferrous iron/mol protein. Using the purification procedure developed for sciatin, we purified a protein from chicken serum that cross-reacted with antisciatin serum, migrated at a position identical to that of sciatin or ovotransferrin on two-dimensional gel electrophoresis, had an amino composition very similar to ovotransferrin and sciatin, and had myotrophic effects on cultured muscle cells. From these data, we conclude that sciatin is a growth-promoting polypeptide closely related in structure to transferrin.     

Evolution of Hawaiian Drosophilidae. II. Patterns and Rates of Chromosome Evolution in an Antopocerus Phylogeny

The phylogenetic relationships of seven species of the genus Antopocerus (Family Drosophilidae) have been determined by means of a study of the metaphase configurations and polytene chromosomes. Based on biogeographical, behavioral and cytogenetic information, A. longiseta from Molokai is tentatively identified as the primitive species of the genus. The metaphase karyotypes of all Antopocerus species are either five pairs of rod chromosomes and a pair of dots (5R1D), or six rods (6R). Heterochromatin additions converted the dots to rods. Chromosome breakpoints for inversions also are clustered at heterochromatic loci. The chromosome segments between heterochromatic loci may represent sets of functionally related loci, evolving as a unit. The rate of chromosomal inversion substitution is estimated in the origin of the taxon (probably a subgenus of Drosophila rather than a separate genus). It averages no greater than one substitution per 1,000 years, or one per 5,000 generations. The average genetic death rate per generation of one individual per hundred is required to achieve this substitution rate. The rate of inversion substitution during radiation of this taxon may be only 4.4 x 10^-3 times as fast as that present in forming the taxon. Alternatively, radiation may have required only 250,000 years if rates of substitution are the same as in the origination of the taxon. Average rates of substitution reflect genetic accidents, selection pressures and rates of adaptation to new niches, as well as the rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Therefore, the average rate of evolution reflects more nearly biogeographic and ecological factors than genetic factors.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

Effects Ala54Thr polymorphism of FABP2 on obesity index and biochemical variable in response to a aerobic exercise training

The purpose of the current study was to investigate whether or not the FABP2 gene polymorphism modulated obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers through 12-week aerobic exercise training in abdominal obesity group of Korean mid-life women. A total of 243 abdominally obese subjects of Korean mid-life women voluntarily participated in aerobic exercise training program for 12 weeks. Polymerase Chain Reaction with Restriction Fragment Length Polymorphism (PCR-RFLP) assay was used to assess the FABP2 genotype of the participants (117 of AA homozygotes, 100 of AT heterozygotes, 26 of TT homozygotes). Prior to the participation of the exercise training program, baseline obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers were measured. All the measurements were replicated following the 12-week aerobic exercise training program, and then the following results were found. After 12-week aerobic exercise training program, wild type (Ala54Ala) and mutant type (Ala54Thr+Thr54Thr) significantly decreased weight (P > .001), BMI (P > .001), %bf (P > .001), waist circumference (P > .001), WHR (P > .001), muscle mass (wild type p < .022; mutant type P > .001), RHR (P > .001), viseceral adipose area (wild type p < .005; mutant type P > .001), subcutaneous area (P > .001), insulin (wild type p < .005; mutant type P > .001) and significantly increased VO2max (P > .001). And wild type significantly decresed NEFA (P > .05), glucose (P > .05), OGTT 120min glucose (P > .05) and significantly increased HDLC (p > .005). Mutant type significantly decreased SBP (P > .001), DBP (P > .01), TC (P > .01), LPL (P > .05), LDL (P > .001), HOMA index (P > .01). The result of the present study represents that regular aerobic exercise training may beneficially prevent obesity index, blood pressure, blood lipids and insulin resistance markers independent of FABP Ala54Thr wild type and mutant type.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Visualization of antibody binding to the photosynthetic membrane: the transmembrane orientation of cytochrome b-559

We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.     

The effect of resistant starch (RS) on the bovine rumen microflora and isolation of RS-degrading bacteria

Applied Microbiology and Biotechnology - Resistant starch (RS) in the diet reaches the large intestine without degradation, where it is decomposed by the commensal microbiota. The fermentation of...