Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.     

Expression of neuronal acetylcholine receptor polypeptidesin vitro

1. Translation of poly(A) RNA extracted from the nervous tissue of locusts in a reticulocyte lysate system led to polypeptides with a broad spectrum of molecular weights. 2. Using anti-locust acetylcholine receptor (AChR) antisera, polypeptides with a molecular weight of about 50,000 were immunoprecipitated. These peptides comprised about 0.3% of the total translation products. 3. Cotranslational incubation with pancreatic rough microsomes resulted in a glycosylated 60,000-dalton immunoprecipitate. 4. Density-gradient analysis of in vitro synthesized and glycosylated receptor polypeptides indicated that no assembly of subunits had taken the place under the in vitro conditions.     

SEM observations on seeds ofStriga spp. andBuchnera americana (Scrophulariaceae)

Seeds of the root parasitesStriga (several spp.) andBuchnera americana were examined by means of SEM. The surface patterns of the seeds in both genera resemble each other closely, especially those ofS. angustifolia andB. americana. SomeStriga spp. can be clearly distinguished by their surface characteristics, while this is quite difficult in others. The taxonomic value of the seed surface features ofStriga andBuchnera is discussed.     

Turn of promotor DNA by cAMP receptor protein characterized by bead model simulation of rotational diffusion

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (less than 100 bp). The final values reflect the solvated structure with the same 'solvation layer' added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix-turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA, provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 A. According to these data the overall bending angle of our longest DNA fragment is approximately 180 degrees, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.     

Induction of unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts treated with cysteine S-conjugates of chlorinated hydrocarbons

S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA aminooxyacetic acid - CEC S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase - DCE 1,2-dichloroethane - DCVC S(1,2-dichlorovinyl)-cysteine - GSH glutathione - HU hydroxyurea - IBR IBR-modified Dulbecco's Eagle's reinforced medium - MN2 micronuclei/2,000 cells - 4-NQO 4-nitroquinoline-1-oxide - SHE Syrian hamster embryo fibroblasts; ³H-Thd, ³H-thymidine - TCE 1,1,2-trichloroethylene - UDS unscheduled DNA synthesis     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Exclusion mapping of the X-linked dominant chondrodysplasia punctata/ichthyosis/cataract/short stature (Happle) syndrome: possible involvement of an unstable pre-mutation

Summary Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata / ichthyosis / cataract / short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (MF) of 1.21 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations.     

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.