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">In Vivo</Emphasis> and <Emphasis Type="Italic">in Vitro</Emphasis> Proliferative and Differentiation Activity of Human Embryonic Retinal Cells

Differentiation of human embryonic retinal cells (20–22 weeks’ gestation) was studied using morphological, immunohistochemical, and biomolecular approaches. The retina included several regions differing by the degree of cell differentiation. Mitoses were rarely found in the marginal zone. This zone contained low differentiated cells. The central retinal area consisted of typical layers with differentiated cells. Culturing was accompanied by the formation of aggregates and neurospheres, where mitoses and progenitor or differentiated cells expressing markers of photoreceptors, neurons, and glia were found.__________Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 103–109, 2005     

Detection and Localization of <Emphasis Type="Italic">Rice stripe virus</Emphasis> Gene Products <Emphasis Type="Italic">In Vivo</Emphasis>

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2–4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N^.and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.     

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">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> evaluation of Bola-surfactant containing niosomes for transdermal delivery

A novel niosome formulation is proposed for topical drug delivery of ammonium glycyrrhizinate, a natural compound with an efficacious anti-inflammatory activity. Niosomes were made up of a new non ionic surfactant, α,ω-hexadecyl-bis-(1-aza-18-crown-6) (Bola-surfactant)-Span 80-cholesterol (2:3:1 molar ratio). Niosome vesicles were prepared with the thin layer evaporation method and were physico-chemically characterized. The tolerability of Bola-surfactant both as free molecules or assembled ion niosome vesicles was evaluated in vitro on cultured of human keratinocyte cells (NCTC2544). Human tolerability was evaluated on volunteers. The ability of Bola-niosomes to promote intracellular delivery was evaluated by confocal laser scanning microscopy (CLSM) studies. Human stratum corneum and epidermis (SCE) membranes were used in vitro to investigate the percutaneous permeation. The anti-inflammatory activity of ammonium glycyrrhizinate was evaluated in vivo on human volunteers with a chemically induced erythema. Experimental data show that Bola-niosomes are characterized by a mean size of ∼400 nm and are able to provide an encapsulation efficiency of 40% with respect to the drug amount used during preparation. CLSM showed that Bola-niosomes were able to promote the intracellular uptake of the delivered substances. Bola-niosomes were also able to significantly improve (p <0.001) the percutaneous permeation of ammonium glycyrrhizinate with respect to both the aqueous drug solution and a physical mixture between unloaded Bola-niosomes and the aqueous drug solution. Bola-niosomes showed a suitable tolerability both in vitro and in vivo. Ammonium glycyrrhizinate-loaded Bola-niosomes determined a significant (p <0.001) and noticeable improvement of the in vivo anti-inflammatory activity of the drug. An effective example of conjugating innovative colloidal carriers, coming from pharmaceutical nanotechnology, and therapeutically effective natural compounds, coming from traditional medicine, was reported. Part of this research was presented at the 33rd Annual Meeting of the Controlled Release Society in Vienna, Austria, July 22–26, 2006.     

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.     

Expression of <Emphasis Type="Italic">GAD67</Emphasis> and Novel <Emphasis Type="Italic">GAD67</Emphasis> Splice Variants During Human Fetal Pancreas Development

Glutamic acid decarboxylase (GAD) is a major inhibitory neurotransmitter in the brain, which catalyses the reaction of l-glutamate to γ-aminobutyric acid. There are two isoforms of GAD, a 65-kDa form and a 67-kDa form, which are encoded by two different genes. As previous studies suggested a role for GAD67 splice variants during fetal pancreas development, we have investigated the mRNA expression of GAD67 and GAD67 splice variants in a series of 14 human fetal pancreases between 14 weeks gestation and term and in adult control pancreases by RT-PCR. In this study, we demonstrate mRNA expression of GAD67 and four GAD67 splice variants, including GAD25, in human fetal and adult specimens. Some of the splice variants, including various proportions of exon 7 or a new exon between exons 6 and 7, have not been described before in the human pancreas. We speculate that the expression of these GAD67 splice variants might be related to human fetal pancreas development.     

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">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.     

PulmoStent: <Emphasis Type="Italic">In Vitro</Emphasis> to <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a Tissue Engineered Endobronchial Stent

Currently, there is no optimal treatment available for end stage tumour patients with airway stenosis. The PulmoStent concept aims on overcoming current hurdles in airway stenting by combining a nitinol stent with a nutrient-permeable membrane, which prevents tumour ingrowth. Respiratory epithelial cells can be seeded onto the cover to restore mucociliary clearance. In this study, a novel hand-braided dog bone stent was developed, covered with a polycarbonate urethane nonwoven and mechanically tested. Design and manufacturing of stent and cover were improved in an iterative process according to predefined requirements for permeability and mechanical properties and finally tested in a proof of concept animal study in sheep for up to 24 weeks. In each animal two stents were implanted, one of which was cell-seeded by endoscopic spraying in situ. We demonstrated the suitability of this membrane for our concept by glucose transport testing and in vitro culture of respiratory epithelial cells. In the animal study, no migration occurred in any of the twelve stents. There was only mild granulation tissue formation and tissue reaction; no severe mucus plugging was observed. Thus, the PulmoStent concept might be a step forward for palliative treatment of airway stenosis with a biohybrid stent device.     

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.     

<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.     

<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.