Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild-type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.     

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Prevalence of duodenal ulcer-promoting gene (dupA) of Helicobacter pylori in patients with duodenal ulcer in North Indian population

BACKGROUND: The duodenal ulcer (DU)-promoting gene (dupA) of Helicobacter pylori has been identified as a novel virulent marker associated with an increased risk for DU. The presence or absence of dupA gene of H. pylori present in patients with DU and functional dyspepsia in North Indian population was studied by polymerase chain reaction (PCR) and hybridization analysis. MATERIALS AND METHODS: One hundred and sixty-six patients (96 DU and 70 functional dyspepsia) were included in this study. In addition, sequence diversity of dupA gene of H. pylori found in these patients was analyzed by sequencing the PCR products jhp0917 and jhp0918 on both strands with appropriate primers. RESULTS: PCR and hybridization analyses indicated that dupA gene was present in 37.5% (36/96) of H. pylori strains isolated from DU patients and 22.86% (16/70) of functional dyspepsia patients (p < or = .05). Of these, 35 patients with DU (97.2%) and 14 patients with functional dyspepsia (81.25%) were infected by H. pylori positive for cagA genotype. Furthermore, the presence of dupA was significantly associated with the cagA-positive genotype (p < or = .02). CONCLUSION: Results of our study have shown that significant association of dupA gene with DU in this population. The dupA gene can be considered as a novel virulent marker for DU in this population.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Molecular mapping of <Emphasis Type="Italic">Rym17</Emphasis>, a dominant and <Emphasis Type="Italic">rym18</Emphasis> a recessive barley yellow mosaic virus (BaYMV) resistance genes derived from <Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.

PK23-2, a line of six-rowed barley (Hordeum vulgare L.) originating from Pakistan, has resistance to Japanese strains I and III of the barley yellow mosaic virus (BaYMV). To identify the source of resistance in this line, reciprocal crosses were made between the susceptible cultivar Daisen-gold and PK23-2. Genetic analyses in the F₁ generation, F₂ generation, and a doubled haploid population (DH45) derived from the F₁ revealed that PK23-2 harbors one dominant and one recessive resistance genes. A linkage map was constructed using 61 lines of DH45 and 127 DNA markers; this map covered 1268.8 cM in 10 linkage groups. One QTL having a LOD score of 4.07 and explaining 26.8% of the phenotypic variance explained (PVE) for resistance to BaYMV was detected at DNA marker ABG070 on chromosome 3H. Another QTL having a LOD score of 3.53 and PVE of 27.2% was located at marker Bmag0490 on chromosome 4H. The resistance gene on chromosome 3H, here named Rym17, showed dominant inheritance, whereas the gene on chromosome 4H, here named rym18, showed recessive inheritance in F₁ populations derived from crosses between several resistant lines of DH45 and Daisen-gold. The BaYMV recessive resistance genes rym1, rym3, and rym5, found in Japanese barley germplasm, were not allelic to rym18. These results revealed that PK23-2 harbors two previously unidentified resistance genes, Rym17 on 3H and rym18 on 4H; Rym17 is the first dominant BaYMV resistance gene to be identified in primary gene pool. These new genes, particularly dominant Rym17, represent a potentially valuable genetic resource against BaYMV disease.     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Eukaryotic phylotypes in aquatic moss pillars inhabiting a freshwater lake in East Antarctica, based on 18S rRNA gene analysis

Aquatic mosses of Leptobryum species form unique tower-like pillars of vegetation termed “moss pillars” in Antarctic lakes. Moss pillars have distinct redox-affected sections: oxidative exteriors and reductive interiors. We have proposed that a “pillar” is a community and habitat of functionally interdependent organisms and may represent a mini-biosphere. Batteries of 16S rRNA genotypes, or phylotypes, of eubacteria and cyanobacteria, but no archaea, have been identified in moss pillars. However, detailed identification or phylogenetic analyses of the moss and their associated eukaryotic microbiota have not been performed. This study analyzed near-full-length 18S rRNA gene sequences obtained from two whole moss pillars. In total, 28 PCR clone libraries from two whole moss pillars were constructed, and 96 clones from each library (total 2,688 clones) were randomly selected and sequenced. Molecular phylogenetic analysis revealed that the phylotype belonging to Bryophyta, considered to be derived from moss, was closely related (99.9?%) to the 18S rRNA gene sequence from Leptobryum pyriforme. Unexpectedly, phylotypes belonging to a novel clade of fungi dominated (approximately 27–75?%) the moss pillar libraries. This suggests that fungi may contribute to carbon cycling in the moss pillar as parasites or decomposers. In addition, phylotypes related to ciliates and tardigrades were subdominant in the exterior, while the phylotype of the ameba-like, single-celled eukaryote, Cercomonas (Cercozoa), was detected only in the interior. These features were shared by both moss pillars. The 18S rRNA gene-based profiles demonstrated that redox-related factors may control distribution of some eukaryotic microbes in a whole moss pillar.