Detection and analysis of extra- and intracellular deoxyribonuclease activities in Lactobacillus plantarum

Extracellular endodeoxyribonuclease activities have been detected in culture supernatant fluids of several Lactobacillus plantarum strains. In the case of Lact. plantarum HER 1325 at least, the extracellular activity is accompanied by a cytoplasmic restriction endonuclease. Among the secreted nucleases, the greatest activity was from Lact. plantarum ATCC 10241. This strain secretes an enzyme, with a molecular mass in the range 10–40 kDa, that cuts double-stranded DNA in a sequence-independent way by initially introducing single-strand nicks in supercoiled molecules, followed by linearization and complete degradation of the substrate.     

Stabilization of a prokaryotic LAT transporter by random mutagenesis

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.     

Effects of central administration of lead, cadmium and other divalent cations on body temperature in mice

The intracerebroventricular administration of lead or cadmium to mice produced a marked decrease in body temperature. The maximum decreases in body temperature produced by the central administration of the heavy metals (1.97±0.3°C for lead and 2.87±0.6°C for cadmium) were similar to those obtained after acute peripheral administration of these compounds. The decreases in body temperature obtained after central administration of calcium, magnesium or zinc were significantly smaller than those produced by the heavy metals. The results indicate that the central nervous system is implicated in the hypothermia induced by these divalent cations and suggest that the alteration of membrane ionic permeability by these substances at hypothalamic sites may be related to the control of body temperature.     

Bone Loss at Implant with Titanium Abutments Coated by Soda Lime Glass Containing Silver Nanoparticles: A Histological Study in the Dog

The aim of the present study was to evaluate bone loss at implants connected to abutments coated with a soda-lime glass containing silver nanoparticles, subjected to experimental peri-implantitis. Also the aging and erosion of the coating in mouth was studied. Five beagle dogs were used in the experiments. Three implants were placed in each mandible quadrant: in 2 of them, Glass/n-Ag coated abutments were connected to implant platform, 1 was covered with a Ti-mechanized abutment. Experimental peri-implantitis was induced in all implants after the submarginal placement of cotton ligatures, and three months after animals were euthanatized. Thickness and morphology of coating was studied in abutment cross-sections by SEM. Histology and histo-morphometric studies were carried on in undecalfied ground slides. After the induced peri-implantitis: 1.The abutment coating shown losing of thickness and cracking. 2. The histometry showed a significant less bone loss in the implants with glass/n-Ag coated abutments. A more symmetric cone of bone resorption was observed in the coated group. There were no significant differences in the peri-implantitis histological characteristics between both groups of implants. Within the limits of this in-vivo study, it could be affirmed that abutments coated with biocide soda-lime-glass-silver nanoparticles can reduce bone loss in experimental peri-implantitis. This achievement makes this coating a suggestive material to control peri-implantitis development and progression.     

Myeloperoxidase-catalyzed chlorination: the quest for the active species

Myeloperoxidase (MPO) is a dominating enzyme of circulating polymorphonuclear neutrophils that catalyzes the two-electron oxidation of chloride, thereby producing the strong halogenating agent hypochlorous acid (ClO⁻/HOCl). In absence of MPO the tripeptide Pro-Gly-Gly reacts with HOCl faster than the amino acid taurine (2-aminoethanesulfonic acid, Tau), while the MPO-mediated chlorination shows reverse order. A comparative study of the enzymatic oxidation of both substrates at pH 4.0–6.0, varying H₂O₂ concentration is presented. Initial and equilibrium rates studies have been carried on, reaction rates in the latter being slower due to the chemical equilibrium between MPO-I and MPO-II–HO₂. A maximum of chlorination rate is observed for Pro-Gly-Gly and Tau when [H₂O₂] ≈ 0.3–0.7 mM and pH ≈ 4.5–5.0. Several mechanistic possibilities are considered, the proposed one implies that chlorination takes place via two pathways. One, for bulkier substrates, involves chlorination by free HOCl outside the heme cavity; ClO⁻ is released from the active center, diffuses away the heme cavity, and undergoes protonation to HOCl. The other implies the existence of compound I–Cl⁻ complex (MPO-I–Cl), capable of chlorinating smaller substrates in the heme pocket. Electronic structure calculations show the size of Pro-Gly-Gly comparable to the available gap in the substrate channel, this tripeptide being unable to reach the active site, and its chlorination is only possible by free HOCl outside the enzyme.     

Rinvenimento del polline di Keteleeria in alcuni depositi quaternari dell'Italia centro-settentrionale

Abstract

THE DISCOVERING OF THE KETELEERIA'S POLLEN-GRAIN IN SOME QUATERNARY DEPOSITS OF THE CENTRAL AND NORTHERN ITALY. — The Author observes the specy of Keteleeria with the pollen analysis executed on deposits of turfys, clays and brown coals of some Umbro-Marchigiano and of the Po Plain localyties.

The Keteleeria to-day is living only in China and Japan; in Italy it was living also during the early and middle Pleistocene.