Analysis and Occurrence of Bisphenol A in Mediterranean Mussels (Mytilus galloprovincialis) Sampled from the Slovenian Coastal Waters of the North Adriatic Sea.

During 2015, a selection of Mediterranean mussels (Mytilus galloprovincialis) and seawater were sampled along the Slovenian coast, which is part of the North Adriatic Sea, in order to test for the presence of bisphenol A (BPA). The concentration of the BPA in the tissues of mussels sampled from the farms and open sea was from < 0.03 to 0.55 µg kg-1 of w.w. and from < 0.03 to 0.46 µg kg-1 of w.w., respectively. The mussel shells from the farms and open sea contained from 0.01 to 0.3 µg BPA kg-1 of w.w. and from 0.04 to 0.27 µg BPA kg-1 of w.w., respectively. In a harbour, mussel tissue and shell concentration of BPA was 0.21 and 0.18 µg BPA kg-1 of w.w., respectively. The seawater in the shellfish farms and the open sea was contaminated with an average of 0.007 µg l-1 of BPA.

Molecular characterisation of noroviruses detected in mussels (Mytilus galloprovincialis) from harvesting areas in Slovenia.

Noroviruses are a leading cause of viral gastroenteritis in humans and are responsible for many outbreaks worldwide. Mussels are one of the most important foodstuffs connected with norovirus outbreaks, also resulting in multinational dimensions. Two hundred and thirty-eight (238) samples of mussels (Mytilus galloprovincialis) were collected in periods between the years 2006-2008 and 2010-2012 to study the prevalence of noroviruses (NoVs) from harvesting areas along the Adriatic coast of Slovenia. Between 2006 and 2008, 9.1% to 24.6% of mussel samples tested by specific GI and/or GII real-time RT-PCR methods were found to be positive for NoVs while between 2010 and 2012 the percentage of NoV positive samples varied from 12.5% to 22.2%. At the nucleotide level within the RdRp gene fragment the genetic diversity of NoVs detected in mussels ranged between 78.8-81.0% nucleotide identity among GII strains (92.1-99.6% within the GII.P4 genotype), 100% nucleotide identity among GI and 58.4-60.2% among GI and GII strains. Nine of the NoV strains detected from mussels were genotyped as GII.4, while two samples were within GI.P2 and one was a positive sample within genotype GII.P21. This study confirmed that mussels are a potential source of the NoV infection. The detected NoVs share the same topology on the phylogenetic tree within the NoV strains detected in water samples and human patients, not only from Slovenia but also from many different countries worldwide. We can assume that mussels in harvesting areas are not only contaminated from the surrounding area but also by contaminated water and sewage from large transport ships, which are regularly present in the area.

Identification of the heparin-binding domain of TNF-alpha and its use for efficient TNF-alpha purification by heparin-Sepharose affinity chromatography.

The N-terminus of the trimeric TNF-alpha molecule comprises two basic arginines within the short amino-acid sequence VRSSSR, which is here shown to be essential for binding of TNF-alpha to heparin-Sepharose. Mixed trimers containing full-length and DeltaN6-truncated subunits revealed a single VRSSSR sequence to be sufficient to achieve binding. On the basis of this newly identified heparin-binding domain, a new method for efficient purification of TNF-alpha is described. Affinity chromatography on heparin-Sepharose was introduced as a key step for highly purified TNF-alpha at a high yield. With minor modifications, this procedure can be used for TNF-alpha analogues that have full-length N-termini, as shown for the less toxic analogue LK-805.

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region.

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan.

Our tumor necrosis factor-alpha (TNF-alpha) analog LK-805 (E107K) exhibited twofold higher specific cytotoxicity on the mouse fibroblast L-929 cell line than its native counterpart. In addition, significantly lowered systemic toxicity was observed in tumor-bearing mouse models treated with this analog. Due to a charge reversal and clustering of three lysines in the exposed tip region of LK-805, we assumed that additional ionic interactions between the positively charged TNF analog and the negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805. To prove this hypothesis, we designed truncated forms of TNF-alpha and analog LK-805 and performed three independent sets of experiments: measurement of cytotoxic activity in the presence of excess heparan sulfate, determination of cytotoxic activity on heparinase-treated L-929 cells, and binding of various TNF-alpha proteins onto the heparin-sepharose affinity column. Cytotoxicity studies of both kinds confirmed the pivotal role of the E107K mutation for interaction with heparan sulfate proteoglycans on the cell surface of L-929 cells. However, heparin-binding studies revealed that intact, full-length N-termini of TNF-alpha or its analogs were necessary for high retention on the heparin affinity column, whereas the three-lysine containing tip of LK-805 by itself was not enough for binding. Obviously, immobilized heparin does not represent an adequate model for membrane-bound heparan sulfate proteoglycans of L-929 cells.

Attachment of histidine tags to recombinant tumor necrosis factor-alpha drastically changes its properties.

When studying two different histidine tags attached to the N-termini of the trimeric cytokine tumor necrosis factor alpha (TNF), the biological activity--measured as cytotoxicity on the L-929 cell line--of both tagged proteins was drastically reduced. The longer His10 tag reduced cytotoxicity to approximately 16% and the shorter His7 tag to 6% of the activity of their nontagged counterparts. After removal of the tags, biological activities reverted to the expected normal values, which clearly shows the key role of the attached histidine tags in diminishing biological activity. Studies on the mechanism of these effects revealed no specific interactions and showed that even the natural flexible N-terminus of TNF presents a steric hindrance for receptor binding, while any extension of the N-terminus increases this hindrance and consequently reduces biological activity. Also, in other proteins, the ligand or substrate binding sites may be hindered by histidine tags, leading to wrong conclusions about biological activity or other properties of the proteins. Thus caution is advised when using His-tagged proteins directly in screening procedures or in research.