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Abstract: The aim of the present study was to investigate the microbial activity along forest brown soil profiles sequence developed on different lithological substrates (carbonate or non-carbonated cement in sandstone formations) at different altitudes. The main question posed was: does carbonate affect the biochemical activity of brown soil profiles at different altitudes? For the purpose of this study, four soil profiles with different amounts and compositions of SOM developed on different lithological substrates were selected: two with carbonate (MB and MZ) and the other two with non-carbonated cement in the sandstone formations (MF1 and MF2). Chemical and biochemical properties of soil were analysed along soil profiles in order to assess the SOM quantity and quality, namely total organic C (C org), water extractable organic C (WEOC) and humification indices (HI, DH, HR). Microbial biomass (C mic and N mic) content, as well as the specific activities of acid phosphatase, β-glucosidase and chitinase enzymes were chosen as indicators of biochemical activity. The soil biochemical properties provided evidence of better conditions for microorganisms in MB than in MF1, MF2 and MZ soil profiles, since patterns of microbial biomass content and activity might be expected in response to the amount and quality of organic substances. The different lithological substrates did not show any clear effect on soil microbial biomass content, since similar values were obtained in MF1, MF2 (with non-carbonated cement) and MZ (with carbonate). However, the specific activities of acid phosphatase (per unit of C org and per unit of C mic) were higher in soils with no carbonate (MF1 and MF2) than in soils with carbonate (MB and MZ). In conclusion, the biochemical activity along brown soil profiles was mainly regulated by different soil organic matter content and quality, while the two different lithological substrates (with carbonate or non-carbonated cement in the sandstone formations) did not show any direct effect on microbial biomass and its activity. However, the activity of acid phosphatase per unit of C was particularly enhanced in soil with non-carbonate cement in the sandstone formations. [Copyright &y& Elsevier]

Changes in carbohydrate and organic acid contents, and the occurrence and potential role of phosphoenolpyruvate carboxykinase (PEPCK) were investigated in gooseberry fruit (Ribes grossularia L., cv. White Smith), in 2006, in central Italy. The concentrations of glucose, fructose, and sucrose (mg g[sup-1] FW) were relatively low up to 60 d after full bloom (DAFB), with glucose and fructose levels being higher than that of sucrose. All then increased up to harvest (approx. 110 DAFB), and sucrose became the most abundant soluble sugar. Malate and citrate concentrations (mg g[sup-1] FW) and contents per fruit (mg fruit[sup-1]) increased up to 70-80 DAFB (malate up to approx. 70 DAFB and citrate up to approx. 80 DAFB) then decreased. The level of PEPCK was determined using both western blotting and enzyme activity measurements. PEPCK increased during fruit ripening and was most abundant when the malate and citrate contents decreased. The results are consistent with PEPCK playing a role in the catabolism of malate and citrate in gooseberry flesh during ripening. [ABSTRACT FROM AUTHOR]

Two drinking water production plants located in North Italy, collecting water from the River Po (Plants 1 and 2) were chosen for this study. Water samples were collected before and after the disinfection process and at two points along the piping system. Water samples were concentrated by the solid-phase extraction system and injected intraperitoneally into specimens of Cyprinus carpio. The concentration of water samples was 3l/equiv. In order to assess the effects of the water samples on carp liver, total glutathione and glutathione-dependent enzymes, such as glutathione S-transferase, glutathione peroxidase, glutathione reductase and glyoxalase I, were measured following this treatment for 6 days at two experimental times (3 and 6 days). Both water plant-treated carp showed a general increase of the enzymatic activities of glutathione S-transferase, and glutathione reductase which might be employed as potential biomarkers of oxidative stress induced by disinfected river water. Plant 1-treated carp showed higher glyoxalase I and glutathione levels and lower glutathione peroxidase activity. A depleted level of total glutathione and of glyoxalase I for specimens of water plant 2 (for both experimental times), without correlation with the distances in the pipeline, suggests that river plant water can also lead to potentially adverse effects on selected biochemical parameters in C. carpio. [Copyright &y& Elsevier]

A seasonal variability of trace metal concentrations and antioxidantenzymes was observed in gills and digestive gland of the Mediterranean mussel Mytilus galloprovincialis from both a polluted and a nonpolluted population. Trace metals (As, Cu, Fe, Mn, Pb, and Zn) exhibited, in both organs, maximum values in later winter--early spring followed by a progressive decrease during the summer. While in the gills this behavior probably reflects a different bioavailability of metals, in the digestive gland it is influenced mainly by the progressive infiltration of the organ by gonadic tissues during gametogenesis. Metals, as other pollutants, are known to influence the oxidative status of these organisms and antioxidant enzymes have been often proposed asbiomarkers of exposure to contaminants. In this respect, it was of interest to compare the variations of these biochemical parameters with those of metal levels in two mussel populations from a polluted anda nonpolluted site, respectively. The biochemical parameters examined included the level of glutathione and the activity of the followingglutathione dependent and antioxidant enzymes: glyoxalase I, EC 4.4.1.5; glyoxalase II, EC 3.1.2.6; glutathione S-transferases, EC 2.5.1.18; glutathione reductase, EC 1.6.4.2; Se-dependent, EC 1.11.1.9 and Se-independent, EC 2.5.1.18 glutathione peroxidases; catalase, EC 1.11.1.6; superoxide dismutase, EC 1.15.1.1. Seasonal variations of trace metals did not appear to influence those of biochemical parameters,which generally showed an opposite trend with higher enzymatic activities in summer when trace metal concentrations were lower. The effects of metals on antioxidant enzymes were more evident when the two mussel populations were compared. In particular, organisms from the polluted site showed lower levels of glutathione and higher enzymatic activities of glyoxalase I even though the magnitude of these differences was not constant during the year. Moreover, native mussels from both th [ABSTRACT FROM AUTHOR]

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease affecting vascular smooth muscle cells of nearly all tissues. Clinical manifestations mainly concern the central nervous system with repeated TIA/stroke, migraine, psychiatric disturbances, and cognitive decline. Minor findings have been reported in muscle, nerve, and skin. CADASIL is due to NOTCH3 gene mutations. This gene has been identified as an up-regulator of c-FLIP, an inhibitor of Fas-ligand-induced apoptosis. The aim of this study was to assess the involvement of oxidative stress-induced apoptosis in cells from 16 Italian CADASIL patients. Peripheral blood lymphocytes (PBLs) and fibroblasts from CADASIL patients were exposed to 2-deoxy-D-ribose (dRib), which induces apoptosis by oxidative stress. Apoptosis was analyzed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy for caspase-3 activation, phosphatidylserine exposure and mitochondrial membrane depolarization. PBLs and fibroblasts from CADASIL patients showed a significantly higher response to dRib-induced apoptosis than those of controls. PBLs from CADASIL patients also showed a significantly higher percentage of apoptotic cells than PBLs from controls, even when cultured without dRib. The greater susceptibility of PBLs and fibroblasts from CADASIL patients to dRib-induced apoptosis suggests that NOTCH3 mutations are an important apoptotic trigger. Since PBLs from patients showed higher levels of apoptosis even in the absence of an apoptotic stimulus, cells from CADASIL patients appear to be physiologically prone to apoptotic cell death.

The endothelial protein C receptor (EPCR) facilitates protein C activation and plays a protective role in the response to Escherichia coli-mediated sepsis in primates. Previously, a soluble form of EPCR (sEPCR) in human plasma was characterized, and several studies indicated that generation of sEPCR is regulated by inflammatory mediators, including thrombin-mediated up-regulation of surface metalloproteolytic activity in vitro. This study addressed the question of whether plasma sEPCR levels reflect changes in thrombin generation in patients undergoing anticoagulant treatment. The sEPCR levels in patients treated with coumarin-type oral anticoagulants were significantly lower than those in healthy asymptomatic adult volunteers (105.3 +/- 70.8 ng/mL [n = 55] versus 165.8 +/- 115.8 ng/mL [n = 200]; P <.0001). A similar decline in plasma sEPCR levels was found in patients treated with unfractionated heparin. In healthy volunteers, sEPCR levels declined to about 100 ng/mL within 3 days after initiation of an 8-day period of warfarin administration and increased within 2 days after its cessation. Plasma sEPCR levels returned to pretreatment values within 1 week, and the changes in plasma sEPCR levels mirrored changes in values for international normalized ratios. A similar decline in sEPCR levels with time was observed in 7 patients beginning treatment with warfarin for a thrombotic disorder. Prothrombin fragment 1 + 2 levels also decreased in volunteers and patients given warfarin. These results show that plasma sEPCR levels decline in response to treatment with anticoagulants whose mechanism of action is known to decrease in vivo thrombin production.

EPCR is a type I transmembrane protein, highly expressed on the endothelium of large vessels, that binds protein C and augments its activation. In this study, a 23bp insertion in the EPCR gene was found in 4/198 survivors of myocardial infarction and 3/194 patients with deep vein thrombosis. The EPCR gene with the insertion predicts a protein that lacks part of the extracellular domain, the transmembrane domain and the cytoplasmic tail. Expression studies showed that the truncated protein is not localized on the cell surface, cannot be secreted in the culture medium, and does not bind activated protein C. Since protein C activation depends on the concentration of EPCR, patients with the EPCR insertion could have a diminished protein C activation capacity. Further clinical studies of adequate samples size are necessary to establish whether or not the EPCR insertion predisposes to the development of thrombotic events.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 kDa and displayed optimal activity at pH 3.0 with a synthetic chromogenic substrate. The N-terminal sequence of this proteinase was determined by automated Edman degradation and was used to design a primer for use in reverse-transcriptase polymerase chain reaction. The open reading frame of the cloned cDNA encoded an aspartic proteinase, which contained the experimentally determined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of kinetic parameters for substrate hydrolysis showed that, at temperatures between 8 and 50 degrees C, the icefish cathepsin D had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50 degrees C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.

To assess whether certain abnormalities of the sulfated amino acid metabolism are associated with the occurrence of thromboembolic events in patients with inherited thrombophilic conditions, the levels of homocyst(e)ine, before or after methionine load, and the presence of the Ala223Val substitution in the 5,10-methylenetetrahydrofolate reductase (MTHFR) were evaluated in 119 subjects with a congenital single thrombophilic condition (type I deficiency of antithrombin n = 10, protein C n = 24, protein S n = 16; activated protein C resistance due to factor V Leiden mutation n = 69). Sixty-three subjects had experienced at least one documented thrombotic event, while the remaining 56 subjects were still free from any thrombotic symptom. Our results show that (1) high homocyst(e)ine levels, either in fasting condition or after methionine load, were not more frequent in subjects with inherited thrombophilic alterations (14.4%) than in normal control subjects (10% by definition) and (2) the frequency of hyperhomocyst(e)inemia was similar in thrombophilic subjects, who already have (14.3%) or have not (14.6%) experienced thrombotic events. As regards the MTHFR mutation, the homozygous condition was present in 23.2% of the thrombophilic patients versus 17.5% in the control subjects, a nonsignificant difference. The mutation was slightly more frequent in those thrombophilic subjects who had suffered a thrombotic episode (25.5%) versus those with no thrombosis (20.8%), with odds ratios of 1.61 (confidence interval (CI) = 0.58-4.52) and 1.24 (CI = 0.42-3.43), respectively. These differences were also nonsignificant. It is concluded that in subjects with inherited thrombophilias, a condition of hyperhomocyst(e)inemia "per se" is not a factor increasing the risk of thrombosis. The risk enhancement conferred by the MTHFR mutation, if any, seems to be slight or limited, and its significance could be ascertained only in a large multicenter trial.

Sixty out of 219 fosfomycin-resistant bacteria selected from more than 7400 urinary pathogens in an epidemiological multicentre survey performed in Italy were screened for plasmid genes fosA and fosB conferring fosfomycin resistance. Only five strains, three enterobacteria and two staphylococci, carried plasmids harbouring, respectively, fosA and fosB genes. Fosfomycin resistance in the other isolates was caused by an alteration of the chromosomally encoded GlpT transport system. One strain, Morganella morganii 279, incorporated alpha-glycerolphosphate and its mechanism of fosfomycin resistance needs to be further investigated. Our study showed that PCR amplification is the most accurate, simple and rapid method for epidemiological studies of plasmid-encoded fosfomycin resistance, and that fosfomycin resistance conferred by plasmid genes (both fosA and fosB) accounts for only a low percentage of the fosfomycin-resistant strains.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in erythrocytes, lymphocytes and muscle of an Italian male, whose family has lived for at least three generations in Lodi (Lombardy, northern Italy). The subject was hospitalized for myalgia and dark urine after intense physical exercise, but no sign of anemia and chronic hemolysis were present at rest. Family studies revealed that the mother and the maternal aunt had the same enzymopathy. The enzyme-specific activity in red blood cells was 15% of control and the kinetic properties were the following: slower electrophoretic mobility; biphasic pH activity curve; slightly reduced thermal stability, and increased utilization of the substrate analogs. The analysis of our patient's DNA showed a G----C mutation at nucleotide 844 which causes an Asp----His amino acid change in position 282. This is the same mutation found by De Vita et al. in the G6PD Seattle-like variant. However, by following a new convention, we labelled our variant as G6PD Lodi844C. As far as the muscle is concerned, we found that the enzyme-specific activity in this tissue was 14% of control values, but cultured myotubes and myoblasts revealed a normal level of G6PD as well as skin fibroblasts. On the contrary in the same type of cultured cells obtained from G6PD Mediterranean subjects, the G6PD activity was about 20% of normal. Our results complete the characterization of this mutant enzyme, demonstrate the expression of the deficit in muscle and describe the enzyme behaviour in cultured cells.