T-cell antigen receptors in Atlantic cod (Gadus morhua l.): structure, organisation and expression of TCR alpha and beta genes

By using short degenerate primers complementing conserved T-cell antigen receptor (TCR) variable and constant region segments for PCR, we were able to isolate putative TCRalpha and beta chain full length cDNAs in Atlantic cod. The Valpha and Vbeta domains have the canonical features of known teleost and mammalian TCR V domains, including conserved residues in the beginning of FR2 and at the end of FR3. The Jalpha and Jbeta region possess the conserved Phe-Gly-X-Gly motif found in nearly all TCR and immunoglobulin light chain J regions. Similar to other vertebrates, the Atlantic cod Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cbeta sequence lacks a cysteine in its connecting peptide region, but other motifs proposed to be important for dimerisation and cell surface expression are observed. Four different cod Cbeta sequences were identified, two of which share 3' untranslated regions different from one of the other two sequences, suggesting the existence of isotypic gene variants of Cbeta. Based on Southern blot analyses, the TCRalpha and beta gene loci appear to be arranged in translocon organisation (as opposed to multicluster) with multiple V gene segments, some (D) and J gene segments and a single or few C gene segments. Northern blot analyses show expression of the TCRalpha and beta chains in thymus, spleen and head kidney, expression of the TCRbeta chain was also detected in the ovary. Interestingly, no expression was detected in intestine even though the existence of T-cells in intestine has been proposed in other teleost species.     

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis.     

Cerebellar cortex and eyeblink conditioning: bilateral regulation of conditioned responses

We examined the role of the cerebellum in classical conditioning of the nictitating membrane response (NMR) of rabbits by comparing the effects of unilateral and bilateral cerebellar cortical lesions. Using extended preoperative conditioning to ensure high levels of learning, we confirmed that unilateral lesions of lobules HVI and ansiform lobe impaired conditioned responses (CRs) previously established to an auditory conditioned stimulus, but did not prevent some relearning with post-operative retraining. Bilateral lesions of HVI and ansiform lobe produced similar impairments of CRs, but also prevented subsequent relearning. Unilateral cortical lesions produced significant enhancement of unconditioned response (UR) amplitudes to periorbital electrical stimulation. Bilateral cortical lesions enhanced UR amplitudes to a lesser extent. Because there was no correlation between the degree of CR impairment and UR enhancement across the unilateral and bilateral lesion groups, the suggestion that the lesions impaired CRs due to general effects upon performance, rather than due to losses of learning, is not supported. Both sides of the cerebellar cortex contribute towards learning a unilaterally trained CR. This finding is important for the re-interpretation of unilateral, reversible inactivation studies that have found no involvement of the cerebellar deep nuclei in the acquisition of NMR conditioning. In addition, we found conditioning-dependent modifications of unconditioned responses that were particularly apparent at low intensities of periorbital electrical stimulation. This finding is important for the re-interpretation of studies that have found apparent changes in the UR of conditioned subjects after cerebellar lesions.     

Evaluation of Etest MBL for detection of blaIMP-1 and blaVIM-2 allele-positive clinical isolates of Pseudomonas spp. and Acinetobacter spp

The Etest MBL (AB BIODISK, Solna, Sweden) correctly differentiated all 57 isolates of Acinetobacter spp. and Pseudomonas aeruginosa with the bla(IMP-1) allele and 135 of 137 (98.5%) Acinetobacter spp. and Pseudomonas spp. isolates with the bla(VIM-2) allele. The Etest MBL was reliable for detecting the IMP-1- and VIM-2-producing Pseudomonas and Acinetobacter isolates.     

Deletion of the ferric uptake regulator Fur impairs the in vitro growth and virulence of Actinobacillus pleuropneumoniae

In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Deltafur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.     

Enteropathogenic Escherichia coli strains carrying genes encoding the PER-2 and TEM-116 extended-spectrum beta-lactamases isolated from children with diarrhea in Uruguay

We studied 13 extended-spectrum beta-lactamase (ESBL)-producing enteropathogenic Escherichia coli isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of blaPER-2 and blaTEM-116. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.     

Site-independent prognostic value of chromosome 9q loss in primary gastrointestinal stromal tumours

Although the significance of tumour site for estimating malignant potential in gastrointestinal stromal tumours (GISTs) has recently been recognized, site-specific genetic patterns have not to date been defined. This study examined 52 c-kit-positive primary GISTs (with a mean follow-up of 42.3 months in 51 cases) from three different locations (35 gastric, 12 small intestinal, and five colorectal) using comparative genomic hybridization (CGH). In general, tumour site correlated with key prognostic factors, including tumour size, mitotic rate, proliferative activity, and probable malignant potential. Furthermore, several DNA copy number changes showed a site-dependent pattern. These included losses at 14q (gastric 83%, intestinal 35%; p = 0.001), losses at 22q (gastric 46%, intestinal 82%; p = 0.02), losses at 1p (gastric 23%, intestinal 88%; p = 1 x 10(-5)), losses at 15q (gastric 14%, intestinal 59%; p = 0.002), losses at 9q (gastric 14%, intestinal 53%; p = 0.006), and gains at 5p (gastric 11%, intestinal 53%; p = 0.002). These data demonstrate strong site-dependent genetic heterogeneity in GISTs that may form a basis for subclassification. Prognostic evaluation of DNA copy number changes identified losses at 9q as a site-independent prognostic marker associated with shorter disease-free survival (p = 0.03) and overall survival (p = 0.002). Furthermore, 9q loss also appeared to carry prognostic value in predicting overall survival for patients with advanced or progressive GISTs (p = 0.003).     

New phenotypic, functional and electrophysiological characteristics of KG-1 cells

Myeloid dendritic cells (DC) are representatives of a rare and phenotypically diverse population of professional antigen presenting cells possessing high functional heterogeneity and flexibility. Here we studied the phenotypic, functional and electrophysiological characteristics of KG-1 cells, an erythroleukemia model cell line, which shares morphological and physiological similarities with immature and mature myeloid DC. We compared the expression of internalizing receptors and other cell surface molecules, antigen uptake and migration of unstimulated and activated KG-1 cells with the characteristics of immature and mature DC. Unstimulated KG-1 cells were less potent in capturing extracellular materials than immature DC. In contrast to monocyte-derived DC KG-1 cells stimulated by PMA and ionomycin ceased to migrate along the MIP-3beta chemokine gradient despite their high expression of CCR7 chemokine receptor and MDR, a transporter implicated in DC migration. Moreover, we determined the ion channel repertoire of KG-1 cells before and after treatment with PMA and ionomycin by using the patch-clamp technique. We found that both unstimulated and activated KG-1 cells expressed time- and voltage-independent, ChTx sensitive intracellular Ca(2+)-gated potassium conductance suggesting the presence of K(Ca) channels in their membranes. Based on our results we propose that KG-1 cells resemble myeloid DC but also possess unique phenotypic, functional and electrophysiological characteristics.