Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.     

Elevation of circulating interleukin-8 is related to lymph node and distant metastases in esophageal squamous cell carcinomas--implication for clinical evaluation of cancer patient

The presence of lymph node metastasis (LNM) is an important factor in clinical evaluation of esophageal cancer patients. Biological markers able to support detection of metastatic lymph nodes are sought after. Interleukin-8 (IL-8) is overexpressed by many cancers and involved in cancer dissemination. We investigated the relationship between circulating IL-8 and clinicopathological features of esophageal squamous cell carcinoma (ESCC), and evaluated the diagnostic potential of IL-8, with reference to the key angiogenic and lymphangiogenic factors: vascular endothelial growth factors A and C (VEGF-A and VEGF-C). We found elevated IL-8 levels in ESCC patients, correlated with tumor size and cancer dissemination, especially LNM. Circulating IL-8 correlated with lymphangiogenic VEGF-C rather then angiogenic VEGF-A. The association weakened in metastatic cancers, suggesting divergent mechanism of IL-8 involvement in the dissemination process. The cytokine levels correlated with platelets and neutrophils, pointing at these cells as possible sources of circulating IL-8. We demonstrated IL-8 that positively correlated with inflammation status of ESCC patients. Circulating IL-8 was a better indicator of ESCC dissemination than VEGF-A or VEGF-C. Yet, the detection rates were not satisfactory enough to allow for the recommendation of IL-8 determination as an adjunct to the clinical evaluation of lymph node involvement in ESCC patients.     

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

A case of Strongyloides stercoralis hyper-invasion in the course of bronchial asthma

A case of Strongyloides stercoralis hyper-invasion is presented. Female patient with the bronchial asthma was periodically treated with steroids. Generalized toxemia caused by parasitic invasion proved fatal in this patient.     

Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis

Using sequence data from the 28S ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of well-accepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is < 40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.      

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.     

Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer.

Basic helix-loop-helix (bHLH) regulatory proteins are known to bind to a single DNA consensus sequence referred to as an E-box. The E-box is present in the regulatory elements of many developmentally controlled genes, including most muscle-specific genes such as troponin I (TnI). Although the E-box consensus is minimally defined as CANNTG, the adjacent nucleotides of functional E-boxes are variable for genes regulated by the bHLH proteins. In order to examine how E-box regulatory regions containing different internal and flanking nucleotides function when placed within the context of a single regulatory element, the E-box region (14 bp) present within the TnI enhancer was substituted with the corresponding E-box sequences derived from the muscle-specific M-creatine kinase (MCK) and cardiac alpha-actin regulatory elements as well as from the immunoglobulin kappa (Ig kappa) enhancer. Within the TnI enhancer, the E-box sequence derived from cardiac alpha-actin was inactive whereas the corresponding sequence from the MCK right E-box efficiently restored wild-type enhancer activity in muscle cells. Intermediate levels of gene activity were observed for TnI enhancers containing E-boxes derived from the MCK left E-box site or from the Ig kappa E2 E-box. DNA binding studies of MyoD:E12 protein complexes with each substituted TnI enhancer confirmed that DNA binding activity in vitro mimics the relative strength of the enhancers in vivo. These studies demonstrate that the specific nucleotide composition of individual E-boxes, which are contained within the regulatory elements of most if not all muscle-specific genes, contributes to the complex regulatory mechanisms governing bHLH-mediated gene expression.