Pseudoquiste traumático de páncreas / Traumatic pseudocyst of the pancreas

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Identification of new isolates of Turnip mosaic virus that cluster with less common viral strains.

Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.

Strains of Turnip mosaic potyvirus as defined by the molecular analysis of the coat protein gene of the virus.

Turnip mosaic virus (TuMV) is a member of the potyvirus genus with a wide host range and highly variable in its biological characteristics. Analysis of the CP gene sequences from databases, combined with the experimental analysis of the CP gene of further isolates, using data derived from sequence or restriction analysis, has allowed the genetic classification of 60 TuMV isolates or sequences. Two main genetic clusters MB (mostly Brassica isolates) and MR (mostly Radish isolates) were found, together with several apparently independent lineages. Isolates in the latter could be grouped as Intermediate between Brassica and Radish clusters (IBR) or outside Brassica and Radish clusters (OBR), according to their genetic distance to the main clusters. The genetic diversity of TuMV isolates deposited in the databases was increased with the sequences of the CP gene of seven selected isolates, mainly belonging to IBR or OBR groups. There was a correlation between the MR genetic cluster and JPN 1 serotype.

Validation of the VITEK2 and the Advance Expert System with a collection of Enterobacteriaceae harboring extended spectrum or inhibitor resistant beta-lactamases.

The susceptibility testing accuracy of the VITEK2 system and the ability of the Advance Expert System (AES) to provide interpretive readings were evaluated against 86 extended spectrum (ESBL) and 6 inhibitor-resistant-TEM (IRT) beta-lactamases producing Enterobacteriaceae clinical isolates. VITEK2 MICs of 12 beta-lactams were compared with those obtained by the standard NCCLS microdilution technique. The overall essential agreement ( +/- 1 log dilution) was 87.8%. Discrepancies were mainly observed with cefepime (30.3% of total number of discrepancies), ceftazidime (21.2%), and cefotaxime (15.1%). MIC discrepancies were slightly higher in CTX-M- (14.4%) than in TEM- (12.5%) or SHV- (11.9%) type ESBL producers and were rare in IRT producers (1.4%). Overall interpretive agreement was 92.5% and minor, major, and very major errors were 5.4%, 1.7%, and 2.1%, respectively. The AES was able to identify an ESBL phenotype in 85 out of 86 isolates (98.8%) and an IRT phenotype in all 6 isolates harboring these enzymes, thus reducing very major errors to 0.9%. The VITEK2 system, in conjunction with the AES software, is a reliable tool for detection of ESBL or IRT producing Enterobacteriaceae isolates.

Characterization of typical pepper-isolates of PVY reveals multiple pathotypes within a single genetic strain.

Potato virus Y (PVY) isolates originally coming from infected pepper plants, were biologically and genetically characterized, especially in comparison with PVY potato-isolates. Pepper PVY isolates could be differentiated from potato isolates in their host range, aphid transmission efficiencies, Mab serology, and genetic status. The genetic distances estimated for PVY pepper-isolates, based on their restrictotypes with five restriction enzymes and on their coat protein gene sequences, indicated that they form a single genetic strain with different pathotypic properties. This situation is essentially different to that of PVY potato-isolates.

The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.

The responses of a collection of Arabidopsis thaliana ecotypes to mechanical inoculation with turnip mosaic potyvirus were assessed. The virus induced characteristic severe symptoms of infection in systemically infected plants. Resistance was found in four ecotypes: Bay-0, Di-0, Er-0, and Or-0. Enzyme-linked immunosorbent assay results of the resistant ecotypes suggested that ecotypes Di-0, Er-0, and Or-0 actually consist of mixed genotypes with resistances acting at different levels in the virus life cycle. Another form of resistance was found in ecotype Bay-0, for which several lines of evidence indicated an interference with viral cell-to-cell movement in the inoculated leaves.

Infectivity of turnip mosaic potyvirus cDNA clones and transcripts on the systemic host Arabidopsis thaliana and local lesion hosts.

Full-length cDNA clones of turnip mosaic virus were assembled under the control of T7 or 35S promoter. The 35S or nopaline synthase terminator signals were introduced downstream the full length cDNA controlled by 35S promoter. Both the capped in vitro transcripts from T7 controlled template, and the cDNAs from 35S controlled plasmids were infectious on Arabidopis thaliana plants according to systemically induced symptoms and to ELISA assays. The cDNAs from 35S controlled plasmids induced local lesions in Chenopodium amaranticolor and Chenopodium quinoa plants. A spontaneous silent C/T transition, giving rise to an additional SpeI restriction site, not present in the original viral RNA template, was used as a marker of the origin of infection.

Potato virus Y group C isolates are a homogeneous pathotype but two different genetic strains.

Potato virus Y group C isolates (PVYC) have been characterized according to biological, molecular and genetic criteria. Two genetic strains, PVYC1 and PVYC2, were identified on the basis of genetic distances (among them and other PVY strains), host range (ability or inability to infect pepper), MAb response (ELISA recognition with MAb 10E3) and coat protein processing site. Some characteristics, such as aphid transmission and ELISA using other MAbs, did not correlate with classification into these two genetic strains. All isolates tested induced a hypersensitive response on potatoes bearing the Nc resistance gene, confirming the nature of PVYC isolates as a homogeneous pathotype.

Amplification and cloning of a long RNA virus genome using immunocapture-long RT-PCR.

A rapid and easy method was developed in order to amplify long fragments of the genome of potato virus Y (PVY), a virus possessing a 10-kb genomic RNA. The method of immunocapture-RT-PCR was adapted, by using thermostable DNA polymerases with proofreading activity and the proper buffers and cycles, to amplify almost the whole genome of PVY in two fragments (5.6 and 4.3 kb) without purifying virions nor viral RNA. Both fragments were cloned subsequently and their ends sequenced. The method is applicable to the rapid cloning and molecular characterization of the genomes of many other RNA viruses.

Modulation of intestinal paracellular permeability by intracellular mediators and cytoskeleton.

The influence of cytoskeletal inhibitors (cytochalasin E and colchicine) and intracellular mediators (cAMP, Ca2+, and protein kinase C) in the control of paracellular permeability to mannitol has been examined in rat jejunum in Ussing-type chambers. Cytoskeletal inhibitors, cytochalasin E (20 mumol.L-1) or colchicine (0.5 mmol.L-1), when present in mucosal, serosal, or in both mediums, significantly increase unidirectional mannitol fluxes. Exposure of mucosal and serosal intestinal surface to 10 mmol.L-1 theophylline or 1 mmol.L-1 cyclic AMP analogue for raising the intracellular cAMP enhances paracellular permeability. In Ca(2+)-free physiological medium passive permeability strongly increases. Alterations of cytosolic Ca2+ levels induced by the Ca2+ ionophore A23187 (20 mumol.L-1) or by 0.3 mmol.L-1 TMB-8, a Ca2+ releasing inhibitor from the intracellular stores, enhance mannitol flux. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C (PKC), also induces a large increase in the intestinal permeability to mannitol. These results provide evidence that tight junctions and consequently epithelial paracellular permeability can be physiologically controlled by intracellular mediators (Ca2+, cAMP, and PKC) probably through modulation of the cytoskeleton activity.

First Report of Grapevine Virus A in Spain.

Grapevine trichovirus A (GVA), a flexuous, filamentous, phloem-limited virus with an approximately 7.3-kbp RNA genome, is widespread in grapevines showing symptoms of leafroll and/or rugose wood. The virus can be mechanically inoculated to Nicotiana benthamiana and N. clevelandii. A field survey of diseased Vitis vinifera white and red cultivars was carried out in Pontevedra (northwest Spain) during the autumn of 1993. We detected the presence of GVA in vines showing leafroll symptoms by an immunocapture-reverse transcription-polymerase chain reaction (PCR) method (2) with GVA-specific primers (1). Bands of the expected size of 430 bp were obtained with extracts from petioles and stem bark as reaction substrates. To verify these results, Northern (RNA) blots with double-stranded (ds) RNAs isolated from grapevines were prepared. Hybridization was positive in two out of 10 samples analyzed. The probe was a 32P-labeled 430-bp PCR product amplified from extracts of N. benthamiana plants infected with GVA strain Is151 (gift of A. Mina-fra). The specificity of this probe was confirmed in dot blot hybridization, as a positive signal was obtained with extracts from GVA-inoculated N. benthamiana, but not with extracts of phosphate buffer-inoculated N. benthamiana, turnip mosaic potyvirus-inoculated Arabidopsis thaliana, or potato potyvirus Y-inoculated N. xanthi plants. The probe did not hybridize to dsRNAs extracted from enzyme-linked immunosorbent assay-positive GLRaV-III-infected grapevines. GVA has been identified in other Mediterranean countries, but to our knowledge this is the first report of the detection of GVA in Spain. References: (1) A. Minafra and A. Hadidi. J. Virol. Methods 47:175, 1994. (2) G. Nolasco et al. J. Virol. Methods 45:201, 1993.

Some technical precisions to a method for in vivo intestinal absorption studies.

Some modifications to the method of PONZ et al. for in vivo intestinal absorption studies using an in situ perfused segment of small intestine, under anesthesia, are described. They improve its accuracy and applicability, especially when slowly absorbed substrates are used or when volume flux measurements are desired. Calculations for the absorbed substrate and net fluid volume change determinations are reported. Several aspects of the use of the method under a single pass or a recirculation perfusion system are discussed.

Effect of osmolarity on the epithelial paracellular permeability in rat jejunum.

Studies in vivo have shown an important increase in the substrate passive permeability across small intestine when Na(+)-cotransported substrates, like galactose, are present at the luminal side. The influence of the solution osmolarity on the passive absorption of mannitol or 2-deoxy-D-glucose across rat jejunum and on the galactose-increasing effect is now studied both in vivo and in vitro. In vivo, luminal perfusion with 400 or 500 mosm/L solutions does not affect passive absorption of 10 mmol/L 2-deoxy-D-glucose, although a net fluid secretion towards lumen is observed. Luminal hyperosmolarity, however, prevents the stimulatory action of 25 mmol/L galactose on the passive absorption, a stimulation that is well manifested in the same intestinal segment when the perfusion is made with isoosmotic solutions. However, in vitro results with everted intestinal sacs or with preparations of intestinal wall in Ussing chambers, indicate that hyperosmolarity (500 mosm/L) of the solutions clearly increases net passive mucosal to serosal flux of D-mannitol or 2-deoxyglucose. With low osmolarity solutions (180 or 160 mosm/L by diminution of NaCl) the in vivo passive mannitol absorption is not affected and the stimulatory action of 25 mmol/L galactose is not observed, although net water absorption is enhanced. Moreover, the passive absorption stimulation by the galactose cotransport in isoosmotic solutions is dependent on Na+ levels requiring at least 80 mmol/L. Results suggest that passive paracellular absorption across the small intestine may be modified by changes in the luminal content composition.

Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana.

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.

A strain-type clustering of potato virus Y based on the genetic distance between isolates calculated by RFLP analysis of the amplified coat protein gene.

Potato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I, Eco RV, Hinf I, Rsa I and Taq I). Genetic distances between the different PVY "restrictotypes" were calculated and used to define the PVY genetic strains. Three main clusters were found: PVYO, PVYN, and non-potato PVY (PVYNP), in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed.

Effect of zinc on D-galactose and L-phenylalanine uptake in rat intestine in vitro.

The effect of zinc on galactose and phenylalanine uptake was studied using rat everted jejunal rings. The rings were incubated for 2 min in oxygenated Krebs-Ringer-Tris (KRT) solution. Galactose and phenylalanine uptake was reduced by zinc in a dose-dependent manner, but not in a time-dependent way. One mM Zn2+ but not 0.5 mM Zn2+ inhibited galactose transport without modifying sugar diffusion. Na(+)-dependent phenylalanine transport was reduced by 0.5 mM and 1 mM Zn2+. However, the metal did not change phenylalanine diffusion obtained in the presence of 40 mM L-methionine or Na(2+)-independent phenylalanine transport. Therefore, zinc seems to interact only with the sodium-galactose or sodium-phenylalanine cotransporters. Zinc inhibited sugar and amino acid transport in a non-competitive way, without a significant change in the affinity of the transporters for their substrates and with a Vmax decrease. The inhibitory effect of Zn2+ on galactose and phenylalanine uptake was reversed by washing intestinal rings for 5 min with KRT solution. These results suggest that zinc might exert its inhibitory action by a weak binding to chemical groups related with sodium-substrate cotransporters and located in the luminal membrane of the enterocytes.