A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Cyanidin-3- O -β-glucopyranoside Protects Myocardium and Erythrocytes from Oxygen Radical-mediated Damages

The cyanidin-3- O - &#103 -glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 &#119 M C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 &#119 M C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 &#119 M C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 &#119 M C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 &#119 M was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 &#119 M). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Dispersione anemofila di polline in Valle Padana

Abstract

Anemophylus dispersal of pollen grains in the Po Valley.—Results of phenological and aerobiological measurements with Castanea sativa pollen are presented. The data concerning the dispersal of this pollen in the Po Valley, show some features of the transport over hundred kilometer distances and give useful suggestions for application research.     

Rendiconti e Atti del Congresso Della Societàg Botanica Italiana a Venezia

Abstract

An investigation has been carried out on the effects of some macroelements on the growth and differentiation of carnation meristem-tip. The meristem-tips were cultured over one year period using BAKER and PHILLIPS (1962) modified medium as basal medium. Different concentration of N, P, K and S were used and their effect on the explants was scored. Variation in the K level had no effect on the growth of the meristems except in one case while P at 1/2 X, 1/4 X to 1/8 X levels considerably increased the number of plantlets obtained. N supplied as urea had an inhibiting action on the explant as two different sources of S did.     

Role of particle coating in controlling skin damage photoinduced by titania nanoparticles

TiO₂ nanoparticles hazard is associated to their photocatalytic activity causing release of DNA damaging ROS (Reactive Oxygen Species), lipid peroxidation and skin damage. Various coatings have been proposed to minimize photocatalysis, while keeping the potential to block UV radiations. Uncoated and variously coated commercial nano-titania have been classified on the basis of UVB-induced lipoperoxidation of linoleic acid. A selection of the most and the least protective specimens was then examined by ESR (Electron Spin Resonance) to evidence the presence of surface paramagnetic centres and the release of ROS in aqueous suspensions (spin trapping). Paramagnetic centres and ROS were correlated with the extent of lipid peroxidation. When tested on porcine skin (mimicking the human one), titania acted as on linoleic acid. The combined use of lipid peroxidation of simple fatty acids with ESR analysis is here proposed as a possible screening tool for the evaluation of the potential toxicity of nano-titania in sunscreen preparations.     

Changes of nitric oxide,carbon monoxide and oxidative stress in term infants at birth

The higher risk of respiratory problem in infants delivered by elective caesarean section in comparison with vaginally born infants may be favoured by lower level of nitric oxide (NO) and carbon monoxide (CO) and higher oxidative stress in infants born by caesarean section. We studied healthy term infants born by vaginal delivery or by elective caesarean section. Nitric oxide, CO, guanosine 3–5 cyclic monophosphate, total hydroperoxide and advanced oxidation protein products (AOPP) were measured at birth and 48–72 h of life. Nitric oxide, CO and cGMP were lower at birth and at 48–72 h of life in infants born by elective caesarean delivery. Total hydroperoxide and AOPP levels were similar in the two groups and increased from birth to 48–72 h of life. In conclusion, nitric oxide and CO concentrations were higher in term infants vaginally born than in infants born by elective caesarean section and decreased from birth to 48–72 h of life. The mode of delivery did not affect the oxidative stress which increases from birth to 48–72 h of life.     

Initial Studies to Define the Physiologic Role of cN-II

IMP preferring cytosolic 5 ′-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5′-inosine monophosphate (IMP), 5′-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy.     

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of ¹⁸F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.     

Evidence for Introduction Bottleneck and Extensive Inter-Gene Pool (Mesoamerica x Andes) Hybridization in the European Common Bean (Phaseolus vulgaris L.) Germplasm

Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L.) in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs) were used to complement chloroplast microsatellite (cpSSRs) and nuclear markers (phaseolin and Pv-shatterproof1) data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from “pure” accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2%) than in America (12.3%). The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with “pure” accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.