Three cases of ophthalmomyiasis externa by sheep botfly Oestrus ovis in Italy

Human infection with the sheep nasal botfly Oestrus ovis is sporadic and is often the consequence of an accidental deposit of the larvae by an adult botfly in the eye. This infestation results in external ophthalmomyiasis that, although a very rare condition, is more common among people living close to farming communities. We report three cases of O. ovis infestation which occurred in Italy in a limited area of La Spezia province (Le Cinque Terre), Italy during summer 2004. None of the patients had contact with wild or farm animals.     

New hypothesis on the role of alternating sequences in calcium-alginate gels

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel.     

Role of the disulphide bridge in folding, stability and function of porcine odorant binding protein: spectroscopic equilibrium studies on C63A/C155A double mutant

Porcine odorant binding protein (pOBP) contains a single disulphide bridge linking residues Cys63 and Cys155. In order to get information on the role played by this crosslink in determining the structural and functional properties of the protein, we substituted these two Cys residues with two Ala residues by site directed mutagenesis and investigated the changes in folding, stability and functional features, as detected by fluorescence and circular dichroism measurements. In particular, we studied both chemical and thermal unfolding/refolding processes under equilibrium conditions, the first induced by guanidinium hydrochloride and the second by raising the temperature from 15 to 90 degrees C. Chemical unfolding curves, as obtained from intrinsic fluorescence and far-UV circular dichroism data, can be fitted by a simple two-state cooperative sigmoidal function; however, their partial overlap (C(1/2)=0.57+/-0.05 from fluorescence and 0.66+/-0.03 from CD) suggests the formation of an intermediate, which lacks tertiary structural features. Thermal unfolding was found to be reversible if the protein was heated up to 65 degrees C, but irreversible above that temperature because of aggregation. The thermodynamic unfolding parameters of this double mutant protein, when compared to those of the wild type protein, clearly point out the important role played by the disulphide bridge on the stability and function of this protein family and probably of many other lipocalins.     

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.     

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions.     

Venous blood lactate evaluation in equine neonatal intensive care

The use of blood lactate concentration as an indicator of prognosis and disease severity has become a common practice in equine medicine, especially with the validation of handheld analyzers. However, few authors described lactate concentration in critically ill foals, and there are no published studies about the use of handheld analyzers in neonatal foals. In this study, for the first time in the equine neonate, we validated the Lactate Scout analyzer, both in healthy and in critically ill foals. The study also describes the normal range for blood lactate in 26 healthy neonatal foals during the first 72 h of life. Moreover, the utility of venous lactate measurement in 88 critically ill foals was determined, describing lactate values in the most common neonatal pathologies, evaluating serial blood lactate measurements, and investigating its prognostic value. The comparison with the enzymatic-colorimetric reference method showed that the Lactate Scout analyzer is reliable. The mean difference (bias ±2SD) between the two methods was close to zero for all comparisons, and the SD of difference was ±0.76 with a 95% confidence interval from −1.58 to 1.40 mmol/L. In healthy foals, blood lactate concentrations at birth and at 12 h of life were statistically higher (P < 0.01) than lactate concentrations measured at subsequent times. In critically ill foals, the highest lactate concentration at admission was found in hemorrhagic shock, septic shock, and complicated perinatal asphyxia syndrome (PAS). Our results showed that hyperlactatemia, although it does not provide diagnostic information, indicates the severity of illness and the need for an early and aggressive intervention. This could be very useful both during hospitalization and in the field to support veterinarians in making a decision about referral. Furthermore lactatemia proved to be a reliable prognostic parameter: In nonsurviving foals, hyperlactatemia persisted during the entire hospitalization, whereas in survivors there were no significant differences after 24 h from admission. Because prognostic parameters have certain limitations, hyperlactatemia should not be used alone to decide whether to discontinue treatments in critically ill foals. A careful and complete clinical examination is always essential.     

Acid gel formation in (pseudo) alginates with and without G blocks produced by epimerising mannuronan with C5 epimerases

The main scope of this paper is the characterization, in terms of viscoelastic and mechanical properties, of acid gels formed from solutions of mannuronan ALG (0%G/0%GG) and its enzymatically epimerised products. The epimerised products were obtained using recombinantly produced mannuronan C5 epimerases named AlgE1 and AlgE4, which catalyse the conversion of mannuronic residues into guluronic (G) and guluronic–mannuronic (GM) blocks, respectively. The products used in this study resulted from either the action of AlgE1 on mannuronan for 5 and 24 h (named ALG(44%G/32%GG) and ALG (68%G/59%GG), respectively) or AlgE4 on mannuronan (named ALG (47%G/0%GG)). d-gluconic acid-δ-lactone (GDL) was used as H⁺-donor to produce acidic gels. ALG (0%G/0%GG) yields strong, stable solid-like structures. As predicted by circular dichroism measurements performed at different pH, gelation of ALG (47%G/0%GG) occurs at lower values of pH (1) than those obtainable using GDL. Hydrochloric acid was therefore added to ALG (47%G/0%GG) solutions yielding rapid sol–gel transitions and gels with a remarkable resistance to thermal treatment.

The introduction of guluronic residues along the chain (ALG (44%G/32%GG)) causes a reduction in the storage modulus at the equilibrium with respect to that of ALG (0%G/0%GG) and the occurrence of negligible syneresis at the highest polymer concentrations. The increase in the average length of the G blocks (ALG (68%G/59%GG)) is accompanied by a further increase in the storage modulus without the occurrence of any significant syneresis.     

Enzymatic cross-linking versus radical polymerization in the preparation of gelatin PolyHIPEs and their performance as scaffolds in the culture of hepatocytes

Highly open porous biodegradable scaffolds, based on gelatin A3, were fabricated with the aim of using them for tissue-engineering applications. The fabrication process is based on an emulsion-templating technique. In the preparation of gelatin scaffolds two different cross-linking procedures were adopted: (I) radical polymerization of the methacrylate functionalities, previously introduced onto the gelatin chains and (II) formation of isopeptide bridges among the gelatin chains promoted by the enzyme microbial transglutaminase. The method of cross-linking exerts a pronounced effect on the morphology of the porous biomaterials: radical polymerization of methacrylated gelatin allowed the production of scaffolds with a better defined porous structure, while the enzymatically cross-linked scaffolds were characterized by a thinner skeletal framework. A suitable sample of each kind of the differently cross-linked porous biomaterials was tested for the culture of hepatocytes. The scaffold obtained by radical polymerization possessed a morphology characterized by relatively large voids and interconnects, and as a consequence, it was more suitable for hepatocytes colonization. On the other hand, the enzymatically cross-linked scaffold resulted in less cytotoxicity and the cultured hepatocytes expressed a better differentiated phenotype, as evidenced by a greater expression and more correct localization of key adhesion proteins.