The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus

A network of circumferentially oriented collagen fibrils exists in the periphery of the human cornea, and is thought to be pivotal in maintaining corneal biomechanical stability and curvature. However, it is unknown whether or not this key structural arrangement predominates throughout the entire corneal thickness or exists as a discrete feature at a particular tissue depth; or if it incorporates any elastic fibres and how, with respect to tissue depth, the circumcorneal annulus integrates with the orthogonally arranged collagen of the central cornea. To address these issues we performed a three-dimensional investigation of fibrous collagen and elastin architecture in the peripheral and central human cornea using synchrotron X-ray scattering and non-linear microscopy. This showed that the network of collagen fibrils circumscribing the human cornea is located in the posterior one-third of the tissue and is interlaced with significant numbers of mature elastic fibres which mirror the alignment of the collagen. The orthogonal arrangement of collagen in the central cornea is also mainly restricted to the posterior stromal layers. This information will aid the development of corneal biomechanical models aimed at explaining how normal corneal curvature is sustained and further predicting the outcome of surgical procedures.     

In vitro assessment of the antimicrobial activity of new N-acyl-thiourea derivatives

The qualitative screening of the susceptibility spectra of different microbial strains to the newly synthesized substances complexes was performed by adapted disk diffusion techniques, while the quantitative assay of the minimal inhibitory concentration (M.I.C., microg/cm3) value was based on liquid medium serial microdilutions. The compounds were solubilized in dimethylsulfoxide (DMSO). The in vitro biological screening effects were tested against a microbial inoculum of approximately 1.5 x 10(8) UFC/cm3, corresponding to 0.5 McFarland standard density, obtained from Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and fungal strains (Candida albicans). In order to investigate the influence of the subinhibitory concentration of the tested substances on the expression of different virulence features, the strains were incubated overnight in the presence of the newly synthesized thiourea derivatives (vol:vol) and different virulence features were investigated, i.e: adherence capacity to the cellular substrate represented by HeLa cells and to inert substrata quantified by slime test and soluble enzymatic virulence factors (haemolysins and other pore-forming toxins, proteases activity, DN-ase and siderophores production). The cytotoxicity was assessed microscopically, by observing the effect of the tested compounds on the cellular substratum integrity.     

Interactions between quercetin and Warfarin for albumin binding: A new eye on food/drug interference

The interaction between quercetin, a popular antioxidant flavonoid, and human serum albumin (HSA) is investigated and characterized by means of induced circular dichroism and saturation transfer difference NMR. These techiques demonstrate the reversible binding of quercetin to the carrier protein, which is responsible for its dissolution in aqueous medium. Competition experiments with two classical probes for HSA binding sites, namely Ibuprofen and Warfarin (a common anticoagulant coumarin), demonstrate that quercetin has a primary binding site located in the subdomain IIA, where coumarins are hosted. The affinity for this site is large and we found that quercetin may effectively displace warfarin from HSA. This may have relevant consequences in rationalizing the interferences of common dietary compounds and food supplements to anticoagulant treatments. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Energy-saving policies and low-energy residential buildings: an LCA case study to support decision makers in Piedmont (Italy)

Background, aim and scope  

A low-energy family house recently built in Northern Italy was selected by Regione Piemonte as an outstanding example of resource efficient building. An economic incentive was awarded to cover the extra costs of the thermal insulation, windows and equipment in order to decrease the yearly winter heat requirement from the legal standard of 109 to 10 kW h/m², while existing buildings in the study area typically require 200 kW h/m². As the building was claimed to be sustainable on the basis of its outstanding energy-saving performance, an ex post life cycle assessment (LCA) was set up to understand whether, and to what extent, the positive judgement could be confirmed in a life cycle perspective.