Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein-disulfide isomerase (PDI)

In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.     

True stress and Poisson's ratio of tendons during loading

Excessive axial tension is very likely involved in the aetiology of tendon lesions, and the most appropriate indicator of tendon stress state is the true stress, the ratio of instantaneous load to instantaneous cross-sectional area (CSA). Difficulties to measure tendon CSA during tension often led to approximate true stress by assuming that CSA is constant during loading (i.e. by the engineering stress) or that tendon is incompressible, implying a Poisson's ratio of 0.5, although these hypotheses have never been tested. The objective of this study was to measure tendon CSA variation during quasi-static tensile loading, in order to assess the true stress to which the tendon is subjected and its Poisson's ratio. Eight equine superficial digital flexor tendons (SDFT, about 30cm long) were tested in tension until failure while the CSA of each tendon was measured in its metacarpal part by means of a linear laser scanner. Axial elongation and load were synchronously recorded during the test. CSA was found to linearly decrease with strain, with a mean decrease at failure of -10.7±2.8% (mean±standard deviation). True stress at failure was 7.1-13.6% higher than engineering stress, while stress estimation under the hypothesis of incompressibility differed from true stress of -6.6 to 2.3%. Average Poisson's ratio was 0.55±0.12 and did not significantly vary with load. From these results on equine SDFT it was demonstrated that tendon in axial quasi-static tension can be considered, at first approximation, as an incompressible material.     

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.     

Skeletal alterations and polymorphism in a Mediterranean bryozoan at natural CO<Subscript>2</Subscript> vents

Colonies of the cheilostome bryozoan Schizoporella errata were grown at a site near Ischia Island (Tyrrhenian Sea, Italy) where volcanogenic CO₂ emissions lower seawater pH to 7.76, simulating levels of ocean acidification predicted for the end of the present century. Compared with colonies from a control site (mean pH = 8.09), putative defensive polymorphs (avicularia) were significantly fewer, and retarded growth of zooidal basal and lateral walls was evident at the low pH site. The lower proportion of avicularia suggests a switch in resource allocation away from defence to favouring rapid growth. In addition, corrosion of the skeleton was observed in both new and old zooids at the low pH site, and feeding zooids were slightly smaller but had larger orifices for the protrusion of feeding lophophores. These findings corroborate previous studies demonstrating potential dissolution of carbonate skeletons in low pH seawater, while providing new insight into the possible ability of colonial species to respond to ocean acidification by adjusting resource allocation between zooids of different types.     

Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic cancer

Despite recognition of the devastating malignant potential of the pancreatic ductal cancer, the exact pathophysiological events contributing to tumor growth remain to be elucidated. Expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were found to be frequently elevated in several types of human cancer and have also been directly linked to carcinogenesis. The purpose of this study was to determine the expression of COX-1, COX-2 and iNOS in human pancreatic cancer and matched normal adjacent tissue by the Western blot assay. Marked COX-2 expression was observed in cancer tissue compared with the normal surrounding tissue. The iNOS protein was markedly expressed only in pancreatic cancer while the expression of COX-1 was similar in both normal and cancerous tissue. Our findings indicate that COX-2 up-regulation and the expression of iNOS in pancreatic cancer, not seen in normal tissue, may play a role in the pathogenesis of human pancreatic adenocarcinomas. These observations suggest that COX-2 and iNOS may be a target for prevention or treatment of pancreatic carcinomas.     

Chronic ethanol treatment: dolichol and retinol distribution in isolated rat liver cells

The aim of this study was to use chronic ethanol intoxication for 2 and 4 months as a means of studying the distribution of dolichol and retinol in isolated rat liver parenchymal cells, Kupffer cells, sinusoidal endothelial cells, and two subfractions of hepatic stellate cells: Ito 1 and Ito 2. Dolichol and retinol were studied in two batches of rats: on normal nutrition and after a load of vitamin A given 3 d before sacrifice. New observations reported are: (i) on normal nutrition, after 2 months of treatment, dolichol in HC seems to be the first target of chronic ethanol, while retinol is the first target in hepatic stellate cells; (ii) the various types of liver cells are differently affected by chronic ethanol, which highlights the importance of studying each type of sinusoidal cell; (iii) a load of vitamin A given when the damage has already occurred restores dolichol content in HC while retinol decreases; and, (iv) a link between dolichol and vitamin A metabolism might be supposed after the load of vitamin A: the percentage distribution of dolichol with 18 isoprene units (Dolichol -18) increases in all the control cells but decreases after chronic ethanol treatment. A different role of this dolichol and/or a different compartmentalization within the cell need to be further investigated.     

Cloning allergens via phage display

Although an impressive list of allergenic structures has been elucidated during the last decade by classical cloning methods, the size of the repertoire of molecular structures able to elicit allergic reactions is still unknown. Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based high-throughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by Escherichia coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures.     

Water-Soluble Ruthenium Complexes Bearing Activity Against Protozoan Parasites

Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl₂(HL)(HPTA)₂]Cl₂ with HL = bioactive 5-nitrofuryl containing thiosemicarbazones and PTA?=?1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl₂(HL4)(HPTA)₂]Cl₂ complex, with HL4?=?N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC₅₀)?=?5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI) >38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites.