Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Metal complexes as artificial proteases in proteomics: a palladium(II) complex cleaves various proteins in solutions containing detergents

Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-[Pd(en)(H₂O)₂]²⁺ (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0% wt./vol. of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 at 2.5 < pH < 2.9 and 55-60 °C for 3-72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.     

Interaction of Trace Elements and Ligninolytic Enzymes in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

Considering the fact that some trace elements have influence on the growth and physiological processes in the fungi, the aim of this study was to resolve the question of whether Fe, Zn, and Se, added to the medium in the different forms, affect activity of selected Pleurotus eryngii ligninolytic enzymes participating in mandarine peels submerged fermentation. Compared with the control, Fe and Zn showed stimulatory effect on laccase activity. Reduction in the activity was emphasized with cultivation time, but it was slower in the presence of Zn than in the presence of Fe. Among studied Se forms, SeO₂ was the most unfavorable Se source for laccase activity. All tested trace elements, independent on form, showed stimulatory effect on the activity of Mn-oxidizing peroxidases on the seventh day of cultivation. Based on the results, it could be concluded that the presence of the tested trace elements can advance various biotechnological processes among which is biodegradation of agricultural wastes, potential environmental pollutants.     

Age,growth and mortality of common two‐banded seabream,Diplodus vulgaris (Geoffroy Saint‐Hilaire, 1817), in the eastern Adriatic Sea (Croatian coast)

Age, growth and mortality were analysed for the common two‐banded seabream, Diplodus vulgaris, collected in the eastern Adriatic (Croatian coast) from commercial fishery catches by ‘tramata’ fishing (2005–2006) to obtain growth estimation. The oldest female was estimated to be age 11, the oldest male age 10 years. The von Bertalanffy growth parameters estimated by reading scales were: L_∞ = 48.60 cm (SE = 1.101), K = 0.112 (SE = 0.005) and t₀ = ?2.366 (SE = 0.060) for all specimens; L_∞ = 51.96 cm (SE = 2.153), K = 0.095 (SE = 0.007) and t₀ = ?2.837 (SE = 0.120) for females and L_∞ = 56.25 cm (SE = 2.662), K = 0.084 (SE = 0.067) and t₀ = ?2.920 (SE = 0.117) for males. The overall sex ratio was 1.22 : 1 in favour of males. Total mortality, corresponding to the slope of the descending limb of the catch curve, was Z = 0.81 per year for females and Z = 0.85 per year for males. Exploitation ratios were E = 0.68 for females and E = 0.73 for males.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

The effect of chronic food restriction on immunopositive ACTH cells in peripubertal female rats

In peripubertal female rats, we have previously found that 50% food restriction (FR) increases plasma IL-6, haptoglobin and both alanine transaminase (ALT) and alkaline phosphatase (AST) aminotransferases, indicating the existence of an inflammatory response. To study whether such FR influences the hypothalamic-pituitary-adrenal (HPA) axis, we examined by immunohistochemistry the morphofunctional features of pituitary adrenocorticotroppic (ACTH) cells. In FR rats the volume and volume density of ACTH cells as well as plasma ACTH levels were increased by 17.6%, 12.5% and 13.4%, respectively, in comparison with controls (p<0.05). We concluded that chronic FR is a systemic stressor in young females, capable to stimulate the HPA axis, probably as a result of IL-6 action.     

Lymphatic vessel invasion detected by the endothelial lymphatic marker D2-40 (podoplanin) is predictive of regional lymph node status and an independent prognostic factor in patients with resected esophageal cancer

The discovery of markers to lymphatic endothelial cells and the development of novel antibodies to these markers have brought increasing attention to the lymphatics and progress in the understanding of lymphangiogenesis and cancer metastasis. In this study, we investigate the presence of lymphatic vessel invasion (LVI) detected by D2-40 immunohistochemical staining in resected esophageal cancer and correlated with clinicopathologic data and patient survival. Sixty nine patients, who had a primary resection of esophageal cancer, were analyzed by univariate and multivariate logistic regression, and univariate and multivariate survival analysis. The total rate of LVI was 72% (50/69). Positive LVI was significantly correlated with lymph node metastasis (p < 0.001), tumor size (p < 0.001), histological grading (p = 0.017), tumor depth (p = 0.001), and stage (p < 0.001). Multivariate logistic analysis identified LVI (p = 0.036) as a predictor of regional lymph node metastasis. On univariate survival analysis, patients with LVI had a significantly shorter disease-free survival, cancer-specific survival and overall survival. Multivariate analysis proved that LVI diagnosed by D2-40 is an independent prognostic factor of both disease-free survival (p = 0.04) and overall survival (p = 0.032) in resected esophageal cancer. These results show that LVI assessment identifies patients at high risk for regional lymph node metastasis and that LVI is an independent prognostic factor in patients with esophageal cancer.     

Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.