Cytokeratin expression in simple epithelia

We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type II) subfamily, which is highly homologous to the corresponding bovine and amphibian (Xenopus laevis) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal 'tail' domain. Comparison with the reported amino acid sequences of two fragments of human 'tissue polypeptide antigen' (TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epithelium-derived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the 'head' portion. These cDNA clones and the RNA probes derived therefrom were used to detect specifically mRNAs by Northern-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.     

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.     

Opioid pharmacology in the rat hippocampal slice

The ability of several opioids in potentiating the synaptic activation of CA1 pyramidal cells in the rat hippocampal slice were compared. Morphine and the opioid peptides, (D-ala2, D-leu5)-enkephalin (DADL), morphiceptin, beta-endorphin, and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSThr) caused a concentration-dependent, naloxone-reversible shift to the left in the input-output (IO) curve constructed by plotting the population spike as a function of the field EPSP. These opioids then produced an increase in the size of the population spike while leaving the EPSP unaffected. In contrast, the kappa agonist prototype, ethylketazocine, had no effect on the IO curve when perfused in concentrations up to 10 microM. The rank order of potency for the opioids in the CA1 region of the hippocampus was DADL greater than DSThr greater than beta-endorphin greater than morphiceptin greater than morphine much greater than ethylketazocine. Thus, opioids that are more specific for delta opiate receptors were the most potent and mu receptor agonists, the least potent in this action. Taken together with previous studies suggesting that morphine and DADL may interact with a common opiate receptor in the CA1 region, the results are consistent with the notion that these epileptiform effects may be primarily mediated by delta opiate receptors in this area although the potency of morphiceptin indicates that mu receptors play some role in this effect.     

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.     

Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas.     

Antibacterial and anti‐inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure‐activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. In this paper, we investigated the antimicrobial and anti‐inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro‐inflammatory chemokines and cytokines interleukin (IL)‐8, IL‐1β, IL‐6 and tumor necrosis factor‐α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro‐inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Functional annotation of genes overlapping copy number variants in autistic patients: focus on axon pathfinding

We have used Gene Ontology (GO) and pathway analyses to uncover the common functions associated to the genes overlapping Copy Number Variants (CNVs) in autistic patients. Our source of data were four published studies [1-4]. We first applied a two-step enrichment strategy for autism-specific genes. We fished out from the four mentioned studies a list of 2928 genes overall overlapping 328 CNVs in patients and we first selected a sub-group of 2044 genes after excluding those ones that are also involved in CNVs reported in the Database of Genomic Variants (enrichment step 1). We then selected from the step 1-enriched list a sub-group of 514 genes each of which was found to be deleted or duplicated in at least two patients (enrichment step 2). The number of statistically significant processes and pathways identified by the Database for Annotation, Visualization and Integrated Discovery and Ingenuity Pathways Analysis softwares with the step 2-enriched list was significantly higher compared to the step 1-enriched list. In addition, statistically significant GO terms, biofunctions and pathways related to nervous system development and function were exclusively identified by the step 2-enriched list of genes. Interestingly, 21 genes were associated to axon growth and pathfinding. The latter genes and other ones associated to nervous system in this study represent a new set of autism candidate genes deserving further investigation. In summary, our results suggest that the autism's "connectivity genes" in some patients affect very early phases of neurodevelopment, i.e., earlier than synaptogenesis.     

Impact of state of arousal and stress neuropeptides on urodynamic function in freely moving rats

Corticotropin-releasing factor (CRF) is a neurotransmitter in Barrington's nucleus neurons. These neurons can coregulate parasympathetic tone to the bladder (to modulate micturition) and brain noradrenergic activity (to affect arousal). To identify the role of CRF in the regulation of micturition, the effects of CRF agonists and antagonists on urodynamics in the unanesthetized rat were characterized. Rats were implanted with bladder and intrathecal or intraperitoneal catheters under isoflurane anesthesia. Cystometry was performed in the unanesthetized, unrestrained state at least 24 h later. In some cases, cortical electroencephalographic activity (EEG) was recorded simultaneously to assess arousal state. During cystometry, the state of arousal often shifted between waking and sleeping and urodynamic function changed depending on the state. Micturition threshold, bladder capacity, and micturition volume were all increased during sleep. The CRF1/CRF2 receptor agonists CRF and urocortin 2 increased bladder capacity and micturition volume in awake but not in sleeping rats. Conversely, the CRF1 receptor antagonists antalarmin and NBI-30775 increased urinary frequency and decreased bladder capacity in awake rats. The present results demonstrate a profound effect of the state of arousal on urodynamic function and suggest that simultaneous monitoring of EEG and cystometry may provide a useful model for studying nocturnal enuresis and other urinary disorders. In addition, the results provide evidence for an inhibitory influence of CRF in the spinal pathway on micturition. Targeting the CRF system in the spinal cord may provide a novel approach for treating urinary disorders.     

Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine.