Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Genetic disruption of mammalian endoplasmic reticulum-associated protein degradation: Human phenotypes and animal and cellular disease models

Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.     

DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

The deleterious effect of stress‐induced depression on rat liver: Protective role of resveratrol and dimethyl fumarate via inhibiting the MAPK/ERK/JNK pathway

This study aimed to uncover the protective potentiality of resveratrol and dimethyl fumarate (DMF) in the liver of a chronic unpredictable mild stress (CUMS)‐induced depression animal model. Resveratrol and DMF significantly alleviated CUMS‐induced behavioral abnormalities in stressed rats through improving sucrose preference in sucrose preference test and decreasing immobility time in a forced swimming test. They also mitigated serum corticosterone levels and elevated serum serotonin levels, which were formerly disturbed in CUMS rats. The hepatoprotective effect is evidenced by improvement in hepatic histopathological examinations, as well as normalized serum alanine aminotransferase and aspartate aminotransferase activities. Molecular signaling of resveratrol and DMF was estimated by diminishing hepatic expression of phosphorylated p38 mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated kinase1/2 (ERK1/2), and c‐Jun N‐terminal kinase (JNK). Consequently, they improved the hepatic antioxidant and anti‐inflammatory activities as elaborated by the normalization of total antioxidant capacity, glutathione, malondialdehyde, nuclear factor‐κB, tumor necrosis factor‐α, and myeloperoxidase levels. In addition, they inhibited hepatocyte apoptosis as evidenced by the increased expression of B‐cell lymphoma 2, the decreased expression of Bax, as well as the suppressed activity of caspase‐3. In conclusion, resveratrol and DMF purveyed a significant anti‐depressant effect, which may be mediated, at least in part, via inhibiting the MAPK/ERK/JNK pathway in the CUMS rat model.     

Induction and Translocation of Tissue Transglutaminase Isoforms Increased Phosphorylation in Retinoic Acid Treated Erythroleukemia Cells

Tissue transglutaminase (TGC, TG2, 80 kDa) is inactive in cross-linking reactions and is converted in vitro and in vivo to the TG (55 kDa) active isoform (Fraij in J Cell Biochem 112:2469–2489, 2011). Two isoforms of human TGC were cloned from human erythroleukemia (HEL) cells induced with retinoic acid (RA) and termed TGH, 63 kDa (Fraij et al. in J Biol Chem 267:22616–22673, 1992) and TGH2, 37 kDa. The purified TGC isoforms exhibited GTPase activity and TGH and TGH2 showed higher activities than the native TGC protein. In all normal cells examined, TGC was found in membrane fractions several fold higher than the supernatant fractions; however, in the natural tumor cell line HEL the TGC cellular distribution was reversed. Although TGC is the major enzyme in normal human erythrocytes, its expression level was significantly decreased in HEL cells. RA treatment induced a sevenfold increase in the level of TGC protein in HEL cells and was accompanied by its translocation to cell membranes. When isolated membrane and supernatant fractions from normal human foreskin (CF3), normal human embryonic lung (WI-38), and HEL cells treated with or without RA were incubated with [³²P]-ATP at 37 °C for 1 h, more radio-labeled proteins were detected in the membrane fractions than the cytosolic fractions. More labeled protein bands were detected in RA treated HEL cells in comparison to control HEL cell extracts. Radio labeled proteins coimmunoprecipitated with the TGC isoforms in RA treated HEL membrane fractions thereby confirming that the radio-labeled material consists of endogenous proteins associated with TGC isoforms. Protein phosphorylation is related to the induction and translocation of the isoforms in RA treated cells. These results show that the TGC isoforms complexes with proteins in vivo and that the phosphorylation of these proteins is catalyzed directly by TGC kinase activity or indirectly by the TGC phosphorylation of other protein kinases.     

Retention in the endoplasmic reticulum is the underlying mechanism of some hereditary haemorrhagic telangiectasia type 2 ALK1 missense mutations

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterised by vascular dysplasia and increased bleeding that affect 1 in 5,000 people world-wide. Pathology is linked to mutations in genes encoding components of the heteromeric transforming growth factor-beta receptor (TGF-beta) and SMAD signalling pathway. Indeed HHT1 and HHT2 result from mutations in the genes encoding endoglin and activin-like kinase 1 (ALK1), TGF-beta receptor components. However, the fundamental cellular defects underlying HHT is poorly understood. Previously using confocal microscopy and N-glycosylation analysis, we found evidence that defective trafficking of endoglin from the endoplasmic reticulum (ER) to the plasma membrane is a mechanism underlying HHT1 in some patients. In this study, we used confocal microscopy to investigate whether a similar mechanism contributes to HHT2 pathology. To do this we expressed wild-type ALK1 and a number of HHT2 patient mutant variants as C-terminally tagged EGFP fusion proteins and tested their localisation in HeLa cells. We found that wild-type ALK1–EGFP was targeted predominantly to the plasma membrane, as evidenced by its colocalisation with the co-expressed HA-tagged endoglin. However, we found that in the majority of cases analysed the HHT2 patient mutant protein was retained within the ER as indicated by their colocalisation with the ER resident marker (calnexin) and lack of colocalisation with cell surface associated HA-endoglin. We conclude that defective trafficking and retention in the ER of mutant ALK1 protein is a possible mechanism of HHT2 in some patients.     

Promoter Analysis for Three Types of EUL-Related Rice Lectins in Transgenic Arabidopsis

In this study, the promoter activity for three types of Euonymus-related lectins (EUL) from rice, further referred to as OrysaEULS2, OrysaEULS3, and OrysaEULD1A was analyzed. In silico promoter analyses showed that the EUL promoters from rice contain next to the typical promoter elements some motifs that are considered to be stress-responsive elements. Furthermore, Arabidopsis thaliana plants were transformed with a promoter::β-glucuronidase (GUS) construct for each of the proteins under study. Subsequently, one-insertion homozygous lines were selected and analyzed for GUS activity. Experiments were performed under normal growth conditions or after application of different stress conditions, in particular treatments with 150 mM NaCl, 100 mM mannitol, and 100 μM abscisic acid (ABA) for 24 h. GUS activity was detected with the OrysaEULS3 and OrysaEULD1A promoters especially in the cotyledons and the young true leaves, respectively, but not with the OrysaEULS2 promoter. The activity of OrysaEULS3 and OrysaEULD1A promoters was increased after ABA and mannitol treatments but decreased after NaCl treatment. We hypothesize that the Euonymus-related rice proteins have a role in sensing and responding to external stresses as well as in the growth of the plant.     

Liquid chromatography “on-flow” 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) 3 sugar residues, and (ii) 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography on-flow proton nuclear magnetic resonance. The LC on-flow NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional ¹H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.     

Preservation of rat skeletal muscle energy metabolism by illumination

Skeletal muscle viability is crucially dependent on the tissue levels of its high energy phosphates. In this study we investigated the effect of the preservation medium Perfadex and illumination with Singlet Oxygen Energy (SOE). Singlet oxygen can be produced photochemically by energy transfer from an excited photosensitizer. The energy emitted from singlet oxygen upon relaxation to its triplet state is captured as photons at 634 nm and is here referred to as SOE. Rat hind limb rectus femoris muscles were preserved for five hours at 22 degrees C in Perfadex, saline, SOE illuminated Perfadex or SOE illuminated saline. Extracts of the muscles were analysed by 31P NMR. Data were analysed using two-way analysis of variance and are given as mean values micromol/g dry weight) +/- SEM. The ATP concentration was higher (p = 0.006) in saline groups (4.52) compared with Perfadex groups (2.82). There was no statistically significant difference in PCr between the saline groups (1.25) and Perfadex groups (0.82). However, there were higher (p = 0.003) ATP in the SOE illuminated groups (4.61) compared with the non-illuminated groups (2.73). The PCr was also higher (p < 0.0001) in the SOE illuminated groups (1.89) compared with the non-illuminated groups (0.18). In conclusion, Perfadex in this experimental model was incapable of preserving the high energy phosphates in skeletal muscle during 5 hours of ischemia. Illumination with SOE at 634 nm improved the preservation potential, in terms of a positive effect on the energy status of the muscle cell.