The canine kallikrein-related peptidase 14: Structural characterization, alternative splicing and differential expression in mammary cancer

Human kallikrein-related peptidases (KLKs) represent a family of 15 serine proteases with diverse roles in many physiological and pathological processes, including carcinogenesis. In the dog, only two KLK genes are known; dKLK1 and canine arginine esterase. Recently, 12 other genes have been predicted using computational methods, but none of them has ever been experimentally validated in canine tissues. In this study we investigated the expression of Canis familiaris KLK14, (CANFA)KLK14, in normal and cancerous mammary tissues. First, it was demonstrated that the in-silico determined canine KLK14 mRNA (GenBank accession no: XM_541464) has been wrongfully predicted on its 5′-end (nucleotides 1–88). The (CANFA)KLK14 mRNA sequence presented here, has high homology to its human counterpart and exhibits all defining-KLK features. In addition to the classical form of the gene, five splice variants were also identified. The splicing events involved 5′-truncation or complete elimination of exon 4 and/or retention of intron I. All encoded protein products of the splice variants were predicted to be truncated and catalytically inactive. The classical form and variant 3 were almost ubiquitously expressed in both normal and neoplastic tissues. Variant 1 was predominantly detected in normal tissues. The classical form and variants 1 and 2 exhibited lower expression levels in tumor compared to normal tissues. Moreover, an Ile155Asn polymorphism was identified. This is the first report on the structural characterization, alternative splicing and tissue expression of canine KLK14 mRNA. These findings may form the basis for the establishment of comparative studies investigating KLK functions in health and disease using the dog as a model.     

Changing social awareness of the illegal killing of migratory birds in the Ionian Islands,western Greece

ABSTRACT

The current study aims at monitoring, measuring and evaluating the ‘Safe havens for wild birds’ campaign, implemented within the framework of the LIFE programme, in Greece and more specifically in the Ionian Islands. The study detects attitude changes that occurred in three target groups (pupils, local hunters and residents) on three islands where the phenomenon of illegal spring killing is more intense. A questionnaire-based survey was conducted in two phases, just before the launch (2013) and after the completion of the campaign (2015), to enable a comparison of answers and data. The results show that pupils and to a lesser extent residents, enhanced their knowledge and awareness of poaching, the migration of avifauna and the consequences of illegal killing on migratory birds, while this aspect of attitude change was not observed in the hunting community which still believes that spring poaching should be treated as a legal activity and part of local culture.     

Characterization of β-amylase alleles in 79 barley varieties with pyrosequencing

β-Amylase is involved in the starch degradation process and therefore influences grain quality. Starch degradation efficiency is dependent on the enzyme thermostability during malting and mashing. Four alleles resulting in different enzyme thermostability are known. These alleles are distinguished by coding single nucleotide polymorphism (cSNP). Pyrosequencing was used for cSNP genotyping of β-amylase alleles in 79 spring barley varieties by using analyser PSQ MA96 System (Pyrosequencing, Biotage). A new cSNP was revealed by means of Pyrosequencing analysis of sequence flanking cSNP⁶⁹⁸, thus recognizing a fifth β-amylase allele. Pyrosequencing is a high-throughput, fast, and precise system for barley SNP genotyping.     

Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA

SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.     

Hsp70 chaperone-based gel composition as a novel immunotherapeutic anti-tumor tool

The recent advances in designing Hsp70-based anti-cancer vaccines and the ability of the chaperone to penetrate inside a living cell prompted us to develop a non-invasive method for the treatment of surface tumors. We designed hydrogel-containing gel-forming substances and human recombinant Hsp70 and applied them on the surface of a 7-day-old B16F10 melanoma tumor. According to the results of histochemistry, Hsp70 diffused through skin layer inside the B16 tumor, and this transport was proved by biochemical data. The application of Hsp70 gel reduced the rate of tumor growth by 64 % and prolonged the life of animals by 46 %. Increased survival was correlated with the enhancement of B16-specific cytotoxicity and up-regulation of gamma–interferon production. Taken together, the data confirm the anti-tumor effect of pure recombinant Hsp70 delivered intratumorally and demonstrate the relevance of a novel non-invasive technology of Hsp70-based therapy.