Urease-aided calcium carbonate mineralization for engineering applications: A review.

Inducing calcium carbonate precipitation is another important function of urease in nature. The process takes advantage of the supply of carbonate ions derived from urea hydrolysis and of an increase in pH generated by the reaction, effects that in the presence of Ca2+ ions lead to the precipitation of CaCO3. Further to its importance in nature, if performed in a biomimetic manner, the urease-aided CaCO3 mineralization offers enormous potential in innovative engineering applications as an eco-friendly technique operative under mild conditions, to be used for remediation and cementation/deposition in field applications in situ. These include among others, the strengthening and consolidation of soil/sand, the protection and restoration of stone and concrete structures, conservation of stone cultural heritage materials, cleaning waste- and groundwater of toxic metals and radionuclides, and plugging geological formations for the enhancement of oil recovery and geologic CO2 sequestration. In view of the potential of this newly emerging interdisciplinary branch of engineering, this article presents the principles of urease-aided calcium carbonate mineralization apposed to other biomineralization processes, and reviews the advantages and limitations of the technique compared to the conventional techniques presently in use. Further, it presents areas of its existing and potential applications, notably in geotechnical, construction and environmental engineering, and its future perspectives.

Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism.

Urease, a Ni-containing metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), K (M) and k (cat), measured at different temperatures and pressures. Such an analysis is useful as it provides information on the molecular nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique in the temperature range 15-35 °C and in the pressure range 5-132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing K (M) provided the characteristics of the formation of the ES complex, and analyzing k (cat), the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic ΔH (o) and ΔS (o) for ES formation, and kinetic ΔH ( ≠ ) and ΔS ( ≠ ) for ES activation, while from the pressure-dependent measurements, the binding ΔV (b) and activation ΔV(#)(cat) volumes were determined. The thermodynamic and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al. (Structure 7:205-216; 1999), in which the Ni-Ni bridging hydroxide--not the terminal hydroxide--is the nucleophile in the catalytic reaction.

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films.

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.

Urease activity and L-ascorbic acid.

In this work, we studied the behaviours of urease in the presence of l-ascorbic acid (AA) and dehydroascorbic acid (DHA) in different conditions. The inactivations of urease were carried out in an unbuffered and buffered system. We show that in the unbuffered system AA inactivated urease in a biphasic manner by denaturation brought about by AA-lowered pH. Further, we show that in the buffered system neither AA nor DHA themselves are inhibitors of urease. The inhibitory action of AA and DHA was revealed in the presence of Fe(3+) ions and most importantly, unlike reported in the literature, it was found to be primarily mediated by H(2)O(2). The resulting inhibition by DHA-Fe(3+) consisted of enzyme thiol oxidation and its effectiveness grew with increasing pH. The results may shed light on the roles of AA in therapies applied in ureolytic bacteria infections, notably those with Helicobacter pylori.

The estimation of oxidative stress markers and apoptosis in right atrium auricles cardiomyocytes of patients undergoing surgical heart revascularisation with the use of warm blood cardioplegia.

Oxidative stress markers and apoptosis were estimated during elective surgical heart revascularization. Eight patients with good ejection fraction underwent coronary artery bypass grafting (CABG) with the use of warm blood cardioplegia. Two right atrium auricle biopsy specimens were collected before and after the operation. Specimens underwent immunocytochemical analysis of mitochondrial manganese superoxide dismutase (MnSOD) expression and apoptosis estimation by the TUNEL method. Ultrastructure analysis under electron microscope was made. Satisfactory results of the operation were obtained. After CABG the MnSOD expression increase in sections of auricles was observed through the increase of stain intensity and the percentage of cells with positive stain (from 30 to 80%). The apoptotic cells percentage remained at approximately the same level. Under the electron microscope insignificant pathological changes were observed. On this basis one may assume that in the case of cardiosurgical procedures with short aorta cross-clamping time and low operation risk level the application of cardioplegia sufficiently prevents reactive oxygen forms (ROF) cytotoxic activity although it does not inhibit the expression of oxidative stress (OS) markers. In our opinion the method of examining right atrium sections is safe and provides results comparable with other publications. It may also be a voice in the discussion on new methods of heart protection during cardiac surgery procedures.

DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

Aggregation of spectrin and PKCtheta is an early hallmark of fludarabine/mitoxantrone/dexamethasone-induced apoptosis in Jurkat T and HL60 cells.

It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKCtheta was associated with spectrin during these changes, suggesting a possible role of spectrin/PKCtheta aggregation in regulation of early apoptotic events. Here we dissect this hypothesis using Jurkat T and HL60 cell lines as model systems. Immunofluorescent analysis of alphaIIbetaII spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKCtheta into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an alphaIIbetaII spectrin fraction that was insoluble in a non-ionic detergent (1% Triton X-100) was observed concomitantly with spectrin aggregation. The changes were observed within 2 h after cell exposure to FND, and preceded PS exposure. The changes seem to be restricted to spectrin and not to other cytoskeletal proteins such as actin or vimentin. In studies of the mechanism of these changes, we found that (i) neither changes in apoptosis regulatory genes (e.g., Bcl-2 family proteins) nor changes in cytoskeleton-associated proteins were detected in gene expression profiling of HL60 cells after the first hour of FND treatment, (ii) caspase-3, -7, -8, and -10 had minor involvement in the early apoptotic rearrangement of spectrin/PKCtheta, and (iii) spectrin aggregation was shown to be partially dependent on PKCtheta activity. Our results indicate that spectrin/PKCtheta aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKCtheta activity. These findings indicate that spectrin/PKCtheta aggregation could be considered as a hallmark of early apoptosis and presents the potential to become a useful diagnostic tool for monitoring efficiency of chemotherapy as early as 24 h after treatment.

Early morphological and functional alterations in canine hepatocytes due to alpha-amanitin, a major toxin of Amanita phalloides.

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of alpha-amanitin (alpha-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of alpha-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme.

The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activity was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg (2+) and Hg2(2+) inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2+ ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2+ binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2+, whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions.

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.

Double mode of inhibition-inducing interactions of 1,4-naphthoquinone with urease: arylation versus oxidation of enzyme thiols.

In their inhibition-inducing interactions with enzymes, quinones primarily utilize two mechanisms, arylation and oxidation of enzyme thiol groups. In this work, we investigated the interactions of 1,4-naphthoquinone with urease in an effort to estimate the contribution of the two mechanisms in the enzyme inhibition. Jack bean urease, a homohexamer, contains 15 thiols per enzyme subunit, six accessible under non-denaturing conditions, of which Cys592 proximal to the active site indirectly participates in the enzyme catalysis. Unlike by 1,4-benzoquinone, a thiol arylator, the inactivation of urease by 1,4-naphthoquinone under aerobic conditions was found to be biphasic, time- and concentration-dependent with a non-linear residual activity-modified thiols dependence. DTT protection studies and thiol titration with DTNB suggest that thiols are the sites of enzyme interactions with the quinone. The inactivated enzyme had approximately 40% of its activity restored by excess DTT supporting the presence of sulfenic acid resulting from the oxidation of enzyme thiols by ROS. Furthermore, the aerobic inactivation was prevented in approximately 30% by catalase, proving the involvement of hydrogen peroxide in the process. When H2O2 was directly applied to urease, the enzyme showed susceptibility to this inactivation in a time- and concentration-dependent manner with the inhibition constant of H2O2 Ki = 3.24 mM. Additionally, anaerobic inactivation of urease was performed and was found to be weaker than aerobic. The results obtained are consistent with a double mode of 1,4-naphthoquinone inhibitory action on urease, namely through the arylation of the enzyme thiol groups and ROS generation, notably H2O2, resulting in the oxidation of the groups.

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Coexpression of CD1a, langerin and Birbeck's granules in Langerhans cell histiocytoses (LCH) in children: ultrastructural and immunocytochemical studies.

Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate the usefulness of immunocytochemical studies on CD 1 a and langerin expression in diagnosis of LCH.

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme.

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ

ABCC2 (MRP2, cMOAT) can be localized in the nuclear membrane of ovarian carcinomas and correlates with resistance to cisplatin and clinical outcome.

PURPOSE: Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells. EXPERIMENTAL DESIGN: For the subcellular detection of ABCC2, immunohistochemistry was done using 41 Federation Internationale des Gynaecologistes et Obstetristes stage III ovarian carcinoma specimens prepared before treatment with cisplatin-based schemes and 35 specimens from the same group after chemotherapy. Furthermore, 11 ovarian carcinoma cell lines as well as tissue microarrays consisting of various human tissues were analyzed. RESULTS: Nuclear membranous localization of ABCC2 was associated with response to first-line chemotherapy at primary (P = 0.0013) and secondary surgery (P = 0.0060). Cases with relapse showed higher nuclear membrane expression at primary (P = 0.0003) and secondary surgery (P = 0.0024). Kaplan-Meier analyses showed that weak nuclear membrane ABCC2 expression before treatment was associated with significantly longer overall (P = 0.04) and progression-free survival (P = 0.001); following chemotherapy, it correlated with significantly longer progression-free survival (P = 0.038). Tissue microarrays confirmed nuclear membranous localization of ABCC2, in particular, in poorly differentiated cells. In ovarian carcinoma cells, it correlated with resistance against cisplatin, whereas localization in the cytoplasmic membrane did not. CONCLUSIONS: ABCC2 confers resistance to cisplatin of ovarian carcinoma in cell culture systems and in clinics when expressed in the nuclear membrane. Thus, ABCC2 localization can predict platinum therapy outcome. Furthermore, expression of ABCC2 in nuclear membranes in human tissues is specific for poorly differentiated cells including stem cells.

Expression of metallothionein in renal tubules of rats exposed to acute and endurance exercise.

The induction of exercise-induced apoptosis in not actively involved in exercise organs, such as kidney could be a result of oxidative stress. Metallothionein (MT) exerts a protective effect in the cell against oxidative stress and apoptosis. We have previously demonstrated an increased incidence of apoptosis in distal tubular cells and collecting ducts in rat kidney after acute exercise. The present study was designed to test the hypothesis that MT may play a protective role in rat renal tubules against exercise-induced apoptosis after the acute exercise and regular training. Male Wistar rats were divided into control, acute exercised and 8-wk regularly trained groups. The kidneys were removed after a rest period of 6 h and 96 h. The ultrastructure of renal tubular cells was examined by electron microscopy. Apoptosis was detected in paraffin sections by the TUNEL technique. Expression of MT was examined by immunohistochemistry. The level of lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was assayed in renal tissue homogenates. After acute exercise, the occurrence of apoptosis was restricted to distal tubules and collecting ducts of rat kidney, whereas the proximal tubules remained unaffected. The 8-wk training did not result in increased apoptosis in tubular cell. MT expression was confined exclusively to proximal tubules in all groups. However, it was significantly increased in acutely exercised animals, as compared to control and trained rats. After the 8-wk training, MT expression remained unaltered as compared to the control group. TBARS levels were significantly increased after acute exercise, while after regular training they remained unchanged. A significant correlation between TBARS level and MT expression was demonstrated. The findings could suggest a protective role of MT against oxidative stress and apoptosis in proximal tubular cells.

[Fatty liver disease--mechanism, clinical significance and factors with a special regard to primary hepatotropic viruses]. / Stluszczenie watroby--patomechanizm, znaczenie kliniczne i czynniki sprawcze, ze szczególnym uwzglednieniem wirusów pierwotnie hepatotropowych.

Fatty liver disease is one of most frequently diagnosed hepatopathies while detailed examination of potential causes of liver enzymes abnormalities is done. Despite its isolated nosology fatty liver disease often co-exists with other liver pathologies or - more often - it is their natural consequence. Liver steatosis is also more often found in primary hepatotropic viral infections. However, it's more prevalent among HCV (hepatitis C virus) infected persons than in those HBV (hepatitis B virus) infected. It is described in 30-70% routinely pursued liver biopsies in HCV infected individuals. Hitherto, there were many analyses concerning clinical and prognostic implications pursued of HCV influence upon inflammatory and fibrotic process of the liver. This article is an up-to-date review of current clinical and therapeutic implications of hepatosteatosis supported by referent study results.

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.

Exercise-induced apoptosis in renal tubular cells of the rat.

A number of studies have shown that acute physical exercise is associated with the induction of apoptosis not only in skeletal muscle but also in many distant organs. One of the pathogenic agents responsible for exercise-induced damage in many tissues is the generation of oxygen free radicals. The aim of the present study was to examine the influence of exercise-induced oxidative stress on the rat kidney. The analysis was performed on the kidneys of rats subjected to treadmill running until exhaustion. Our results demonstrated that acute exercise led to apoptotic damage of the renal distal tubular cells, although this was not a result of oxidative stress.