Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.     

Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.     

LUCAS - A New LCIA Method Used for a Canadian-Specific Context

Goal, Scope and Background Canadian LCA practitioners currently use European or American methodologies when conducting comprehensive impact assessments, despite the fact that these methods may not be appropriate for Canadian conditions. Due to the lack of suitable models that are currently available, work has been undertaken to develop an LCIA method by adapting existing LCIA models to the Canadian context. This new method allows the characterization of 10 impact categories. Methods This project is strongly based on preliminary outcomes from SETAC recommendations for the best available practices in LCIA. Models from 3 recent LCIA site-dependent methods, EDIP2003, IMPACT2002+ and TRACI, were used in this midpoint Canadian-specific method. Characterization models were chosen based on their level of comprehensiveness, scientific sophistication and the possibility of integrating site-specific values in the models. Results and Discussion All regional and local impact categories in the method are site-differentiated. For aquatic eutrophication, (eco)toxicity and land-use impact categories, regionally-differentiated models taking into account fate and effect were already available: the parameters of these models were modified for the Canadian context. For acidification, aquatic and terrestrial eutrophication, existing models were spatially differentiated for fate: regionalization of the effect factor was also included, based on the level of sensitivity of each ecozone assessed with vulnerability factors. The default spatial resolution selected for this method was Canadian ecozones, which define spaces in an ecologically meaningful way where organisms and their physical environment evolve as a system. For each ecozone, 2334 site-dependent characterization factors have been calculated. Conclusion This LCIA methodology proposes an attractive and useful set of site-dependent characterization factors for the 15 Canadian terrestrial ecozones. Recommendation and Outlook Efforts are being carried out to extend the specificity of some factors used in eutrophication modelization. Finally, the transparency of the methodology will allow to re-calculate site-dependent characterization factors for different regions and for additional substances.     

UVC radiation-induced effect on human primary thyroid cell proliferation and HLA-DR expression

The aim of this study was to examine how UVC irradiation will affect normal human thyroid cell proliferation and HLA-DR expression. Primary human thyroid cells were exposed to UVC (254 nm wavelength) irradiation. In some experiments 0.5 mM buthionine sulfoximine (BSO) was added. Apoptosis was detected measuring annexin V, proteins involved in apoptotic process (p53, Bax, Bcl-2, caspase 3, and 9) by immunoblot analysis and HLA-DR expression by FACS. UVC induced a cell cycle arrest in G0/G1 phase in the first 24 h, accumulation of cells in the S phase 72 h after treatment, and an increase of apoptotic cells. BSO pretreatment showed an earlier appearance and a higher percentage of apoptosis. p53, caspase 3 and 9 were increased, while Bax and Bcl-2 were decreased. We also observed a transient significant increase in HLA-DR expression. UVC inhibited cell proliferation and induced apoptosis in normal human primary thyroid cells. An inhibitor of glutathione synthesis induced an earlier appearance and higher percentage of apoptosis suggesting that oxidative stress may play a role. Apoptotis involved components of the intrinsic mitochondrial pathway. A transient increase in HLA-DR expression after UVC irradiation could play a role in inducing AITD.     

Tsg101 Interacts with Herpes Simplex Virus 1 VP1/2 and Is a Substrate of VP1/2 Ubiquitin-Specific Protease Domain Activity

Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme.     

Circulating stem cell vary with NYHA stage in heart failure patients

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45⁻CD34⁻CD90⁺, CD45⁻CD34⁻CD105⁺ and CD45⁻CD34⁻CXCR4⁺ were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45⁻CD34⁻CD90⁺CXCR4⁺cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4⁺ subpopulation of HSC (CD45⁺CD34⁺CD90⁺CXCR4⁺, 1.4 versus 13.3 cells/μl in controls and NYHA class III patients, respectively) and TCSC (CD45⁻CD34⁺CXCR4⁺, 1.5 cells/ μl in controls versus 12.4 and 28.6 cells/μl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90⁺ expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.     

Brain Endo-Oligopeptidase A, a Putative Enkephalin Converting Enzyme

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.     

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.