Characterization of SrgA,a Salmonella enterica serovar Typhimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA,essential for biogenesis of plasmid-encoded fimbriae

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.     

Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.     

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.     

Effects of 2-benzoxazolinone on the germination, early growth and morphogenetic response of mung bean (Phaseolus aureus)

2‐Benzoxazolinone (BOA), a type of hydroxamic acid present in cereals and implicated in allelopathy, is now being viewed as a potential candidate for the development of natural herbicides. A study was conducted to determine the effect of BOA on mung bean (Phaseolus aureus) through a multitude of bioassays to understand its physiological and biochemical action. It was observed that BOA significantly decreased the germination of mung bean and its early growth (measured in terms of seedling length and dry weight). A typical dose–response relationship was observed with BOA treatment, and I₅₀ values (concentrations at which 50% inhibition occurs) for germination, seedling length and seedling dry weight were calculated to be 4.3, 0.71 and 0.77 mM , respectively. There was therefore a greater inhibitory effect on seedling growth than on germination. Treated seedlings were characterised by a loss of chlorophyll and decreased respiratory activity, indicating a possible adverse effect of BOA on photosynthetic and respiratory metabolism. Mitotic activity in root‐tip cells of onion (Allium cepa) was completely arrested in response to BOA treatment, and the cells exhibited abnormality in shape and size. BOA also adversely affected rhizogenesis in hypocotyl cuttings of mung bean, indicating an impact on morphogenetic potential. It was associated with significant changes in the protein content and activities of proteases and polyphenol oxidases during the root development phase. This study concludes that BOA interfered with essential biochemical processes in mung bean. Such studies provide useful information on the biochemical and physiological modes of actions of BOA, with a view to its use as a herbicidal compound.     

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses.     

Effect of low voltage on electrical properties of eye lens

In the present study, the Cole-cole plot of lens tissue has been drawn using AC impedance system (EG and G PARC Model 318) in the frequency range 10 mHz to 10 Hz at low voltage stress. The impedus locus between real part (Z') and imaginary part (Z") of complex impedance of lens was examined. Results showed that the extracellular resistance (Re), distribution factor (alpha) and depressed angle (theta) were significantly varied at experimental low voltages. An attempt has been made to explain the electrical data of voltage-tissue interaction on the basis of solid state biophysics.     

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.     

Over-marking and Adjacent Marking are Influenced by Sibship in Male Prairie Voles,Microtus ochrogaster

Scent over-marking occurs when an animal deposits its scent mark on top of the scent mark of a conspecific. Over-marking may provide advantages in the transfer of information to the individual whose scent is on top but not to the individual whose scent is on the bottom. We tested the hypothesis that over-marking is a competitive form of olfactory communication and that male prairie voles would over-mark the scent marks of same-sex conspecifics more than those of same-sex siblings. Two age-matched male voles (first male and second male) were placed successively into an arena in which they were allowed to explore freely and scent mark for 15 min at age 12, 20, 28, 36, 44, and 52 d. The first male was placed into a clean arena, whereas the second male was placed into an arena containing either the scent marks of an age-matched male sibling or nonsibling. Age affected the total number of scent marks deposited by the voles; 12-20-d-old voles deposited fewer scent marks, over-marks and adjacent marks than did 28-52-d-old voles. Sibship did not affect the total number of scent marks deposited by the first and second voles but did affect the number of over-marks and adjacent marks deposited by the second vole. Siblings received significantly fewer over-marks and adjacent marks than did nonsiblings; this effect was most dramatic after the voles reached 28 d of age, a time coincident with the onset of puberty. Males separated from siblings and housed singly at 44-d-old and tested at 52-d-old, deposited significantly more over-marks and adjacent marks in arenas if the first vole was a nonsibling than if it was a sibling. This differential scent-marking supports the hypothesis that over-marking and adjacent marking are used as competitive forms of olfactory communication by male prairie voles.     

Molecular analysis of the genome of transgenic rice (Oryza sativa L.) plants produced via particle bombardment or intact cell electroporation

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.     

Medical housing "lines".

General practitioners are often asked for medical certificates (housing "lines") by applicants for council housing who claim to have medical problems requiring housing priority. The results of a survey by questionnaire showed that general practitioners in Edinburgh do not know how the housing system works and that they seem to overestimate their patients'' chances of obtaining suitable council housing. General practitioners need to know how the housing system works, and communication between general practitioners and housing departments should be improved. A comparison was also made between the number of medical points awarded by a community medicine specialist and a group of general practitioners who had written housing "lines" for their patients. The general practitioners tended to award more points than the specialist. Social priority for housing should be recognised as an independent factor and a new category of top social priority added.