Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

Behaviour of <Emphasis Type="Italic">Sinapis alba</Emphasis> chromosomes in a <Emphasis Type="Italic">Brassica napus</Emphasis> background revealed by genomic <Emphasis Type="Italic">in-situ</Emphasis> hybridization

Genomic in-situ hybridization (GISH) was applied to study the behaviour of addition chromosomes in first and second backcross (BC) progenies of hybrids between Brassica napus ssp. napus L. (AACC, 2n = 38) and Sinapis alba L. (SS, 2n = 24) produced by electrofusion. With GISH using genomic DNA of S. alba was used as probe it was possible to clearly distinguish both of the parental genomes and effectively monitor the fate of S. alba chromosomes in the BC₁ and BC₂ progenies. GISH analysis confirmed the sesquidiploid genome composition (AACCS) of the BC₁ progenies, which contained 38 chromosomes from B. napus and 12 chromosomes from S. alba. Genome painting in the pollen mother cells (PMCs) of the BC₁ plants revealed intergenomic association between B. napus and S. alba chromosomes, whereby a maximum of 4 trivalents between AC and S chromosomes were identified at metaphase I. In the BC₂ progenies, aneuploids with different numbers of additional chromosomes from S. alba, ranging from 1 to 7, were confirmed. Three putative monosomic alien addition lines were characterized, and the results are discussed with respect to the potential for intergenomic chromosome recombination.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Effect of bioactive fractions of <Emphasis Type="Italic">Citrullus vulgaris</Emphasis> Schrad. leaf extract against <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Aedes aegypti</Emphasis>

The benzene extract of Citrullus vulgaris was tested against Anopheles stephensi and Aedes aegypti for the larvicidal activity and ovicidal properties. The crude benzene extract was found to be more effective against A. stephensi than A. aegypti. The LC₅₀ values were 18.56 and 42.76 ppm respectively. The LC₅₀ values for silica gel fractions (bioactive fractions I, II, III and IV) were 11.32, 14.12, 14.53 and 16.02 ppm respectively. The mean per cent hatchability of the egg rafts were observed after 48 h post treatment. The crude extract of benzene exerted 100% mortality at 250 ppm against A. stephensi and at 300 ppm against A. aegypti. The silica gel fractions I and II afforded 100% mortality at 100 ppm and III and IV exerted the hatchability rate of 4.9 and 5.3% at the same concentration against A. stephensi.     

Tracing origin of serrated adenomas with <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">KRAS</Emphasis> mutations

Serrated neoplasm of the colorectum raised many as-yet unanswered issues. To characterize serrated neoplasia pathway, we investigated BRAF and KRAS mutations in 35 traditional serrated adenomas. BRAF exons 11 and 15, and KRAS exon 2 were amplified by polymerase chain reaction and directly sequenced. BRAF V599E mutation was found in 27 serrated adenomas (77.1%), and KRAS mutations were found in 3 (8.6%) of 35 traditional serrated adenomas. In 13 cases, mixed polyps composed of traditional serrated adenomas and hyperplastic (serrated) polyps were observed, and seven of them showed the same BRAF mutations in both components. Somatic mutations of BRAF and KRAS genes were mutually exclusive. These findings suggest that BRAF mutations are early and a critical event in the serrated adenomas, and most serrated adenomas in both sides of colon may progress from microvesicular hyperplastic polyps via BRAF mutations, and some left-sided serrated adenomas develop via KRAS mutations.     

Age-Dependent Stability of Sensorimotor Functions in the Life-Extended <Emphasis Type="Italic">Drosophila</Emphasis> mutant <Emphasis Type="Italic">Methuselah</Emphasis>

Methuselah is a Drosophila mutant with a 35% increased lifespan. We examined the robustness of methuselah’s sensorimotor abilities in tethered flight as a function of age in experiments designed to test visuomotor synchronization and phototaxis in simulated flight. A total of 282 flies from different age groups (4 hours to 70 days) and genotypes (mth and w1118) were individually tethered under an infrared laser-sensor system that digitally recorded wing-beat frequency (WBF). We found that mth has a higher average WBF throughout most of its lifespan compared to parental control flies (w1118) and develops flight ability at a younger age. Its WBF at late life, however, is not significantly different than that of its parental control line. We further found that mth entrains during flight to motion of a visual grating significantly better than its parental line. These findings suggest that the mth gene not only delays chronological aging but enhances sensorimotor abilities critical to survival during early and middle, but not late life. Edited by Yong-Kyu Kim     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.     

Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

<Emphasis Type="Italic">Prospero</Emphasis> Mutants Induce Precocious Sexual Behavior in <Emphasis Type="Italic">Drosophila</Emphasis> Males

Brain maturation, a developmental process influenced by both endogenous and environmental factors, can affect sexual behavior. In vertebrates and invertebrates, sexual maturation is under the influence of hormones and neuromodulators, but the role of developmental genes in this process is still poorly understood. We report that prospero (pros), a gene crucial for nervous system development, can change the age of onset of sexual behavior in Drosophila melanogaster males: adult males carrying a single copy of several pros mutations court females and mate at a younger age than control males. However, these pros mutations had no effect on female sexual receptivity and did not alter other male phenotypes related to mating behavior. The Pros protein was detected in several brain and sensory structures of immature adult males, some of which are normally involved in the regulation of male specific behaviors. Our data suggest that the altered pros expression affects the age of onset of male mating behavior.     

Expression of <Emphasis Type="Italic">HSPF1</Emphasis> and <Emphasis Type="Italic">LIM</Emphasis> in the lymphoblastoid cells derived from patients with bipolar disorder and schizophrenia

We have previously reported the altered expressions of HSPF1 and LIM in the lymphoblastoid cell lines (LCLs) derived from Japanese patients with bipolar disorder (bipolar I disorder). The altered expression at the LCL level would be useful for developing diagnostic markers as well as a cellular model for bipolar disorder. In this study, we extended our previous study by measuring their expressions using the following samples: (1) larger number of LCLs from Japanese subjects, (2) LCLs from Caucasian subjects, and (3) LCLs from patients with bipolar II disorder or schizophrenia. We confirmed the increased expression of HSPF1 (P=0.009) and decreased expression of LIM (P=0.001) in the LCLs from patients with Japanese bipolar I disorder. These altered expressions were also observed in those from patients with Japanese bipolar II disorder (P=0.002 for HSPF1 and P=0.072 for LIM). We also found the altered expressions of HSPF1 in LCLs from Caucasian patients with bipolar II disorder (P=0.011) and LIM in those from patients with schizophrenia (P=0.001).     

A chitinase gene, <Emphasis Type="Italic">chiB</Emphasis>, involved in the autolytic process of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Chitinases are thought to be involved in the morphogenesis and autolysis of filamentous fungi. We cloned a gene (chiB) encoding a class V chitinase from Aspergillus nidulans. ChiB expressed in Escherichia coli had chitin-hydrolyzing activity, indicating that chiB encoded a chitinase. Deletion of chiB affected neither germination efficiency nor hyphal growth rate, but considerably reduced the intracellular and extracellular chitinase activities. The decrease in hyphal dry weight during autolytic phase was slower in the mutant than in the wild-type strain. Western blot analysis indicated that the quantity of ChiB significantly increased when the wild-type mycelia were starved for carbon sources, a condition that induced hyphal autolysis. These results suggest that chiB plays an important role in the autolytic process in A. nidulans.