Modelling intracellular H(+) ion diffusion

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.     

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.     

Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.     

Changes in dietary sodium consumption modulate GLUT4 gene expression and early steps of insulin signaling

Previous studies have shown that chronic salt overload increases insulin sensitivity, while chronic salt restriction decreases it. In the present study we investigated the influence of dietary sodium on 1) GLUT4 gene expression, by No the n and Western blotting analysis; 2) in vivo GLUT4 protein translocation, by measuring the GLUT4 protein in plasma membrane and microsome, before and after insulin injection; and 3) insulin signaling, by analyzing basal and insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-beta, insulin receptor substrate (IRS)-1, and IRS-2. Wistar rats we e fed no mal-sodium (NS-0.5%), low-sodium (LS-0.06%), o high-sodium diets (HS-3.12%) fo 9 wk and were killed under pentobarbital anesthesia. Compared with NS ats, HS ats inc eased (P < 0.05) the GLUT4 protein in adipose tissue and skeletal muscle, whereas GLUT4 mRNA was increased only in adipose tissue. GLUT4 expression was unchanged in LS ats compared with NS ats. The GLUT4 translocation in HS ats was higher (P < 0.05) both in basal and insulin-stimulated conditions. On the other hand, LS ats did not increase the GLUT4 translocation after insulin stimulus. Compared with NS ats, LS ats showed reduced (P < 0.01) basal and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle and IRS-2 in live, whereas HS ats showed enhanced basal tyrosine phosphorylation of IRS-1 in skeletal muscle (P < 0.05) and of IRS-2 in live. In summary, increased insulin sensitivity in HS ats is elated to increased GLUT4 gene expression, enhanced insulin signaling, and GLUT4 translocation, whereas decreased insulin sensitivity of LS ats does not involve changes in GLUT4 gene expression but is elated to impaired insulin signaling.     

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.     

Identification and genetic characterisation of orthopaedic Staphylococcus isolates collected in Italy by automated EcoRI ribotyping

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis, both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph system for 96% of the isolates. All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson's index of discrimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among isolates with the identical ribotyping profile.