Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl- into the shoots

Eight wild Hordeum species: H. bogdanii, H. intercedens, H. jubatum, H. lechleri, H. marinum, H. murinum, H. patagonicum, and H. secalinum, and cultivated barley (H. vulgare) were grown in nutrient solution containing 0.2 (control), 150, 300, or 450 mol m(-3) NaCl. In saline conditions, the wild Hordeum species (except H. murinum) had better Na+ and Cl- 'exclusion', and maintained higher leaf K+, compared with H. vulgare. For example, at 150 mol m(-3) NaCl, the K+:Na+ in the youngest, fully expanded leaf blades of the wild Hordeum species was, on average, 5.2 compared with 0.8 in H. vulgare. In H. marinum grown in 300 mol m(-3) NaCl, K+ contributed 35% to leaf psi(pi), whereas Na+ and Cl- accounted for only 6% and 10%, respectively. By comparison, in H. vulgare grown at 300 mol m(-3) NaCl, K+ accounted for 19% and Na+ and Cl- made up 21% and 25% of leaf psi(pi), respectively. At 300 mol m(-3) NaCl, glycinebetaine and proline together contributed almost 15% to psi(pi) in the expanding leaf blades of H. marinum, compared with 8% in H. vulgare. Decreased tissue water content under saline conditions made a substantial contribution to declines in leaf psi(pi) in the wild Hordeum species, but not in H. vulgare. A number of the wild Hordeum species were markedly more salt tolerant than H. vulgare. H. marinum and H. intercedens, as examples, had relative growth rates 30% higher than H. vulgare in 450 mol m(-3) NaCl. Hordeum vulgare also suffered up to 6-fold more dead leaf material (as a proportion of shoot dry mass) than the wild Hordeum species. Thus, several salt-tolerant wild Hordeum species were identified, and these showed an exceptional capacity to 'exclude' Na+ and Cl- from their shoots.     

Lco1 is a novel widely expressed lamin-binding protein in the nuclear interior

A-type lamins are localized at the nuclear envelope and in the nucleoplasm, and are implicated in human diseases called laminopathies. In a yeast two-hybrid screen with lamin C, we identified a novel widely expressed 171-kDa protein that we named Lamin companion 1 (Lco1). Three independent biochemical assays showed direct binding of Lco1 to the C-terminal tail of A-type lamins with an affinity of 700 nM. Lco1 also bound the lamin B1 tail with lower affinity (2 microM). Ectopic Lco1 was found primarily in the nucleoplasm and colocalized with endogenous intranuclear A-type lamins in HeLa cells. Overexpression of prelamin A caused redistribution of ectopic Lco1 to the nuclear rim together with ectopic lamin A, confirming association of Lco1 with lamin A in vivo. Whereas the major C-terminal lamin-binding fragment of Lco1 was cytoplasmic, the N-terminal Lco1 fragment localized in the nucleoplasm upon expression in cells. Furthermore, full-length Lco1 was nuclear in cells lacking A-type lamins, showing that A-type lamins are not required for nuclear targeting of Lco1. We conclude that Lco1 is a novel intranuclear lamin-binding protein. We hypothesize that Lco1 is involved in organizing the internal lamin network and potentially relevant as a laminopathy disease gene or modifier.     

Involvement of integrins and Src in insulin signaling toward autophagic proteolysis in rat liver

Cell volume changes critically determine hepatic signal transduction and metabolism. Hepatocyte swelling by insulin contributes to p38(MAPK) activation leading to inhibition of autophagic proteolysis. Recently integrins were shown to sense hypoosmotic hepatocyte swelling. Here the role of integrins, Src, and focal adhesion kinase (FAK) in insulin signaling was investigated using the intact organ model of perfused rat liver. Insulin increases [Tyr(P)(418)]Src, [Tyr(P)(397)]FAK, and dual p38(MAPK) phosphorylation by about 2-fold. Infusion of the integrin-antagonizing hexapeptide GRGDSP or the Src inhibitor PP-2 prevented activation of Src and p38(MAPK) and, consequently, proteolysis inhibition by insulin. However, insulin-induced phosphorylation of IRbeta (Tyr(1158)) and protein kinase B (PKB, Ser(473)), as well as K(+)-uptake and cell swelling, was not reduced by the inhibitors. Both hypoosmotic swelling and insulin increase the plasma membrane levels of activated beta(1) integrin. Inhibition of insulin-induced swelling by furosemide largely abolished activation of beta(1) integrin and phosphorylation of Src, but not of PKB. Rapamycin does not affect either insulin-induced K(+)-retention and cell swelling or proteolysis inhibition, indicating that swelling-dependent proteolysis inhibition occurs independently from the mammalian target of rapamycin. The data suggest that sensing of cell swelling by integrins essentially contributes to insulin signaling, thereby defining a novel way of integrin involvement in growth factor signaling.     

Channel-forming (Porin) activity in Herpetosiphon aurantiacus Hp a2

Detergent extracts of cell envelopes of the gliding bacterium Herpetosiphon aurantiacus formed channels in lipid bilayers. Fast protein liquid chromatography across a HiTrap-Q cation-exchange column demonstrated that a 45-kDa protein forms the channel. The observation of a channel-forming protein suggests that Herpetosiphon aurantiacus Hp a2 has a permeability barrier on its surface.     

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.     

Mating-type locus control of killer toxins from Kluyveromyces lactis and Pichia acaciae

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.     

Identification of conjugated linoleic acid elongation and beta-oxidation products by coupled silver-ion HPLC APPI-MS

Atmospheric pressure photoionisation (APPI) was used in combination with silver-ion (Ag(+))-HPLC for detection of (conjugated) fatty acid methyl esters (FAME) by tandem-mass spectrometry. APPI-MS of methyl esters of conjugated linoleic acid showed an increase in signal-to-noise ratio by a factor of 40 compared to atmospheric pressure chemical ionization in the positive mode. It was possible to identify double bond position, configuration and chain length of FAME based on chromatographic separation and mass detection. The developed LC-MS method is useful for the analysis of CLA elongation and beta-oxidation products, especially with trans,trans-configuration, which are difficult to analyze by conventional GC-MS techniques.     

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.     

DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.