The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

TREES SIFTER 1.0: an approximate method to estimate the time to the most recent common ancestor of a sample of DNA sequences

trees sifter 1.0 implements an approximate method to estimate the time to the most recent common ancestor (TMRCA) of a set of DNA sequences, using population evolution modelling. In essence, the program simulates genealogies with a user‐defined model of coalescence of lineages, and then compares each simulated genealogy to the genealogy inferred from the real data, through two summary statistics: (i) the number of mutations on the genealogy (M_n), and (ii) the number of different sequence types (alleles) observed (K_n). The simulated genealogies are then submitted to a rejection algorithm that keeps only those that are the most likely to have generated the observed sequence data. At the end of the process, the accepted genealogies can be used to estimate the posterior probability distribution of the TMRCA.     

Fluid replacement following dehydration reduces oxidative stress during recovery

To investigate the effects of hydration status on oxidative DNA damage and exercise performance, 10 subjects ran on a treadmill until exhaustion at 80% VO_2max during four different trials [control (C), 3% dehydration (D), 3% dehydration + water (W) or 3% dehydration + sports drink (S)]. Dehydration significantly decreased exercise time to exhaustion (D < C and S). Plasma MDA levels were significantly higher at pre-exercise in D than C. Plasma TAS was significantly lower at pre-exercise in C and S than in D, and was significantly lower in S than D at 60 min of recovery. Dehydration significantly increased oxidative DNA damage during exercise, but fluid replacement with water or sports drink alleviated it equally. These results suggest that (1) dehydration impairs exercise performance and increases DNA damage during exercise to exhaustion; and (2) fluid replacement prolongs exercise endurance and attenuates DNA damage.     

Unusual DNA binding modes for metal anticancer complexes

DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophobic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes.     

Modulation of DNA precursor pools, DNA synthesis, and ultraviolet sensitivity of a repair-dificient CHO cell line by deoxycytidine

The presence of 2 mM deoxycytidine (CdR) in growth medium substantially increased the deoxycytidine triphosphate (dCTP) and deoxuthymidine triphosphate (dTTP) pools in a Chinese hamster ovary cell line, CHO-K1, and in a radiation-sensitive mutant, xrs-5, derived from it (Jeggo et al., 1982). We observed significant differences in alkaline-sucrose gradient profiles of pulse-labeled DNA from unirradiated CHO-K1 and xrs-5 cells. For the latter cell line, a sizable fraction of the DNA synthesized during 5 or 10 min of growth subsequent to a 5-min radiolabeling period was found to co-sediment with large-chromosome DNA. This characteristics of xrs-5 was dramatically reduced by the presence of 2 mM CdR in the culture medium, and the UV resistance of the mutant increased to nearly that of the parent cell line under these culture conditions. These results show that the locus conferring UV-radiation sensitivity to xrs-5 affects DNA replication and that replicative activity and UV-radiation sensivity are jointly modulated by CdR, possibly through its impact on the size of deoxynucleoside triphosphate pools.     

Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis

Abstract The molecularly cloned gene encoding the vesicular stomatitis virus (VSV) membrane glycoprotein G was modified and joined to a Bacillus subtilis secretion vector constructed from the plasmid pUB110 and containing the promoter and signal sequence regions of the α-amylase (a secretory protein) gene from Bacillus amyloliquefaciens . The regions encoding the NH₂-terminal signal peptide and the COOH-terminal hydrophobic transmembrane domains of the VSV gene were deleted to facilitate the secretion of the G protein in soluble form. The truncated G protein was found to be expressed in B. subtilis . The expression level was low, probably due to rapid proteolytic degradation of the protein and, contrary to what was expected, almost all of the protein remained cell-associated.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two‐step PCR protocol enabling the “all at once” taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis‐assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost‐effective screening tool for addressing evolutionary ecological issues or developing “chirosurveillance” and conservation strategies.