Hypermethylation of<Emphasis Type="Italic"> Chfr</Emphasis> and<Emphasis Type="Italic"> hMLH1</Emphasis> in gastric noninvasive and early invasive neoplasias

Human tumors are genetically unstable, and the instability exists at two distinct levels—the chromosomal level and the nucleotide level. Chfr and hMLH1 hypermethylation, which may lead to chromosomal instability (CIN) and microsatellite instability (MSI), respectively, was analyzed in gastric noninvasive neoplasias (NIN, Padova international classification) and submucosal invasive adenocarcinomas and in their corresponding non-neoplastic gastric epithelia. Results were compared with microsatellite status, p53 immunoreactivity, and cellular phenotype. Hypermethylation of Chfr and hMLH1 was observed in: 10% (1/10) and 0% (0/10) of low-grade NIN (L-NIN); 63% (5/8) and 63% (5/8) of high-grade NIN, including suspicion for carcinoma without invasion (H-NIN); 36% (5/14) and 57% (8/14) of high-grade NIN, including carcinoma without invasion; and 35% (7/20) and 25% (5/20) of submucosal invasive adenocarcinomas, respectively. Hypermethylation was less frequent in L-NIN than H-NIN (P<0.05) for Chfr and was also less frequent in L-NIN than the others (P<0.05) for hMLH1. We failed to find a significant correlation between Chfr hypermethylation and chromosomal loss of heterozygosity, although hypermethylation of hMLH1 was significantly associated with high-frequency MSI (P<0.01). Expression of p53 was not associated with Chfr or hMLH1 methylation. As for cellular phenotype, hypermethylation of Chfr and hMLH1 was frequent in tumors exhibiting the foveolar epithelial phenotype (50%, 2/4 and 75%, 3/4, respectively) and the ordinary phenotype (40%, 16/40 and 38%, 15/40, respectively), but never in those with the complete-type intestinal metaplastic phenotype (0%, 0/8 for both). In addition, hypermethylation of Chfr and hMLH1 occurred concurrently (P<0.01); methylation was more frequent in patients over 70 years of age (P<0.01), and it was also present in some samples of non-neoplastic gastric epithelia from elderly patients. Thus, some gastric tumors with the foveolar or ordinary phenotype may develop as a result of age-related methylation of Chfr and hMLH1, although Chfr methylation was not associated with CIN.     

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.     

A subset of colorectal carcinomas express c-KIT protein independently of <Emphasis Type="Italic">BRAF</Emphasis> and/or <Emphasis Type="Italic">KRAS</Emphasis> activation

c-KIT is a tyrosine kinase receptor found to be overexpressed in several tumours, namely, GISTs, breast, lung, prostate, ovarian and colorectal carcinomas (CRC). We aimed at determining the frequency of c-KIT expression and mutations in a series of 109 CRC cases (73 primary tumours and 36 lymph node metastases) characterised for KRAS and BRAF mutations. We also aimed at analysing the cellular effects of STI571/Gleevec in CRC-derived cell lines displaying c-KIT expression and KRAS or BRAF mutations. By immunohistochemistry, we found c-KIT overexpression in 15% (11/73) of primary tumours and in 14% (5/36) of metastasis; however, cases showing overexpression did not show c-kit mutations in hotspot regions. The majority (64%) of primary tumours with c-KIT overexpression had mutations at KRAS–BRAF genes. The same was true for 60% of the metastases. We treated CRC cell lines with STI571/Gleevec and verified that it inhibits proliferation and induces apoptosis in all cell lines. In conclusion, overexpression of c-KIT is observed in a subset of primary and CRC metastases in the absence of c-kit mutations. STI571/Gleevec increases apoptosis in CRC cell lines independently of its genetic profile, suggesting that STI571/Gleevec is likely to be an alternative drug for the clinical trials of CRC.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

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.     

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.     

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.     

<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.     

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.     

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.     

<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.     

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">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.     

<Emphasis Type="Italic">MECP2 </Emphasis>and <Emphasis Type="Italic">CDKL5</Emphasis> gene mutation analysis in Chinese patients with Rett syndrome

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.