Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: Insight into the molecular mechanism of transport

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coli, purified and reconstituted in liposomes, and their transport activity was measured as ³H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.     

Use of desalting gel for the rapid separation of simple sugars from exopolysaccharides produced by lactic acid bacteria

Three methods to remove simple carbohydrates prior to the measurement of exopolysaccharide concentration with the phenol/sulphuric acid method were compared. A new method based on size exclusion chromatography on a desalting gel compared favourably with ethanol precipitation (which was simple and rapid, but resulted in underestimation of EPS concentration) and dialysis (which was long and cumbersome). © Rapid Science Ltd. 1998     

Effect of ursodeoxycholic and chenodeoxycholic acid therapy in patients with cholesterol gallstones on intestinal microflora and its bile-acid-metabolizing activity

The in vitro transformation of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), and 7-keto-lithocholic (6-keto-LCA) acid by fecal specimens from five patients with cholesterol gallstones, treated with UDCA and CDCA, and five healthy control subjects was compared. Degradation of CDCA, UDCA, and 7-keto-LCA to lithocholic acid (LCA) was generally faster in fecal cultures of treated patients than in those of controls; this finding correlates with the significantly greater number of microorganisms found to be able to produce LCA from both CDCA and UDCA. Comparative analysis of intestinal microflora composition in the two groups indicates that only the number of bifidobacteria, Gram-positive anaerobic cocci, and coliforms is increased in patients compared with normal, untreated subjects.     

Pharmacokinetic and immunogenic behavior of three recombinant human GM-CSF-EPO hybrid proteins in cynomolgus monkeys

MEN 11300, MEN 11301, and MEN 11303 are three recombinant human hybrid proteins that, as has recently been described, induce in vitro erythroid differentiation. This article provides data on their pharmacokinetic and immunogenic behavior after repeated iv administration to cynomolgus monkeys at 0.8 or 1.6 μg/kg doses. Pharmacokinetic data, obtained after the first administration, showed that the half-life (t _1/2) and clearance (CL) values are dose dependent, with no significant differences among the three hybrid proteins. After the tenth administration, MEN 11300 and MEN 11301, both at high and low dose, and MEN 11303 at high dose were undetectable in plasma, whereas MEN 11303 at the lower dose showed no alteration in its pharmacokinetic profile. Immunologic analyses of plasma provided an explanation for this different pharmacokinetic behavior. In fact, plasma samples from animals treated repeatedly with MEN 11300 and MEN 11301 showed specific antibody formation in response to both the high- and the low-dose regimens. These antibodies exerted in vitro a strong neutralizing activity of the hybrid proteins, with a predominant specificity for the erythropoietin (EPO) portion. By contrast, MEN 11303 at the lower dose did not induce a detectable antibody response whereas the antibodies observed on the high-dose regimen did not exert neutralizing activity against the hybrid proteins nor against granulocyte-macrophage colony-stimulating factor (GM-CSF) or EPO. Hematologic parameters were not affected by the treatments, thus indicating that the anti-EPO neutralizing antibody response does not cross react with the endogenous monkey cytokine. The overall immunogenicity data suggest that among the three fusion proteins, MEN 11303 could have a lower immunogenic potential.     

Pathogenicity variation in Fusarium verticillioides populations isolated from maize in northern Italy

One hundred and eighty one strains were selected among Fusarium verticillioides populations isolated from maize samples collected in three fields located in northern Italy. All the isolates were tested for their pathogenicity on maize seeds by assessing the seed germination percentages and the percentage infection indexes concerning seed colonization, radicle decay and coleoptile rot. Fusarium verticillioides strains did not affect seed germination even in presence of high seed colonization, but showed a variable pathogenic behavior according to the maize growth stages. Seedborne F. verticillioides population as well as strains isolated at maturity was effective in seed colonization and in inducing coleoptile rot, not causing however serious radicle decay. Only populations isolated at seedling and pre-silking stages showed high radicle decay ability. These results provide baseline information on F. verticillioides pathogenicity. They constitute an important input for further investigation of F. verticillioides biology in order to define its evolutionary potential.     

A New Weighted Degree Centrality Measure: The Application in an Animal Disease Epidemic

In recent years researchers have investigated a growing number of weighted heterogeneous networks, where connections are not merely binary entities, but are proportional to the intensity or capacity of the connections among the various elements. Different degree centrality measures have been proposed for this kind of networks. In this work we propose weighted degree and strength centrality measures (WDC and WSC). Using a reducing factor we correct classical centrality measures (CD) to account for tie weights distribution. The bigger the departure from equal weights distribution, the greater the reduction. These measures are applied to a real network of Italian livestock movements as an example. A simulation model has been developed to predict disease spread into Italian regions according to animal movements and animal population density. Model’s results, expressed as infected regions and number of times a region gets infected, were related to weighted and classical degree centrality measures. WDC and WSC were shown to be more efficient in predicting node’s risk and vulnerability. The proposed measures and their application in an animal network could be used to support surveillance and infection control strategy plans.