The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABC DEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wildtype and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

Calibration of the channel that determines the omega-hydroxylation regiospecificity of cytochrome P4504A1 - Catalytic oxidation of 12-halododecanoic acids

The fatty acid omega-hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in (H2O)-O-18 show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O-2. The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an omega-1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the omega-aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[H-2](2)-12-chlorododecanoic acid causes a 2-3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl >> I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 angstrom but smaller than 4.30 angstrom.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) - a breakdown product of haem with cytoprotective and toxic properties - is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5-10 h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC50 approximately equal to the substrate concentration. The 7-methoxyresorufin 7-O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

Urinary porphyrins as biomarkers for arsenic exposure among susceptible populations in Guizhou Province, China

Coal is widely used in PR China. Unfortunately, coal from some areas in Guizhou Province contains elevated levels of arsenic. This has caused arsenicosis in individuals who use arsenic-contaminated coal for the purposes of heating, cooking and drying of food in poorly ventilated dwellings. The population at risk has been estimated to be approximately 200,000 people. Clinical symptoms of arsenicosis may include changes of skin pigmentation, hyperkeratosis of hand and feet, skin cancers, liver damage, persistent cough and chronic bronchitis. We analyzed the porphyrin excretion profile using a HPLC method in urine samples collected from 113 villagers who lived in Xing Ren district, a coal-bome arsenicosis endemic area and from 30 villagers from Xing Yi where arsenicosis is not prevalent. Urinary porphyrins were higher in the arsenic exposed group than those in the control group. The correlation between urinary arsenic and porphyrin concentrations demonstrated the effect of arsenic on heme biosynthesis resulting in increased porphyrin excretion. Both uroporphyrin and coproporphyrin III showed significant increases in the excretion profile of the younger age (< 20 years) arsenic-exposed group, suggesting that porphyrins could be used as early warning biomarkers of chronic arsenic exposure in humans. Greater increases of urinary arsenic and porphyrins in women, children and older age groups who spend much of their time indoors suggest that they might be at a higher risk. Whether elevated porphyrins could predict adverse health effects associated with both cancer and non-cancer end-points in chronically arsenic-exposed populations need further investigation. (c) 2005 Elsevier Inc. All rights reserved.

Complexes of cytotoxic chelators from the dipyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

In an effort to better understand the antiproliferative effects of the tridentate hydrazone chelators di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and di-2-pyridyl ketone benzoyl hydrazone (HPKBH), we report the coordination chemistry of these ligands with the divalent metal ions, Mn, Co, Ni, Cu, and Zn. These complexes are compared with their Fe-II analogues which were reported previously. The crystal structures of Co(PKIH)(2), Ni(PKIH)(2), Cu(PKIH)(2), Mn(PKBH)(2), Ni(PKBH)(2), Cu(PKBH)(2), and Zn(PKBH)(2) are reported where similar bis-tridenate coordination modes of the ligands are defined. In pure DMF, all complexes except the Zn-II compounds exhibit metal-centered M-III/II (Mn, Fe, Co, Ni) or M-II/I (Cu) redox processes. All complexes show ligand-centered reductions at low potential. Electrochemistry in a mixed water/DMF solvent only elicited metal-centered responses from the Co and Fe complexes. Remarkably, all complexes show antiproliferative activity against the SK-N-MC neuroepithelioma cell line similar to (HPKIH) or significantly greater than that of the (HPKBH) ligand which suggests a mechanism that does not only involve the redox activity of these complexes. In fact, we suggest that the complexes act as lipophilic transport shuttles that allow entrance to the cell and enable the delivery of both the ligand and metal which act in concert to inhibit proliferation.

Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase

The sulfite dehydrogenase from Starkeya novella is the only known sulfite-oxidizing enzyme that forms a permanent heterodimeric complex between a molybdenum and a heme c-containing subunit and can be crystallized in an electron transfer competent conformation. Tyr236 is a highly conserved active site residue in sulfite oxidoreductases and has been shown to interact with a nearby arginine and a molybdenum-oxo ligand that is involved in catalysis. We have created a Tyr236 to Phe substitution in the SorAB sulfite dehydrogenase. The purified SDHY236F protein has been characterized in terms of activity, structure, intramolecular electron transfer, and EPR properties. The substituted protein exhibited reduced turnover rates and substrate affinity as well as an altered reactivity toward molecular oxygen as an electron acceptor. Following reduction by sulfite and unlike SDHWT, the substituted enzyme was reoxidized quickly in the presence of molecular oxygen, a process reminiscent of the reactions of the sulfite oxidases. SDHY236F also exhibited the pH-dependent CW-EPR signals that are typically observed in vertebrate sulfite oxidases, allowing a direct link of CW-EPR properties to changes caused by a single-amino acid substitution. No quantifiable electron transfer was seen in laser flash photolysis experiments with SDHY236F. The crystal structure of SDHY236F clearly shows that as a result of the substitution the hydrogen bonding network surrounding the active site is disturbed, resulting in an increased mobility of the nearby arginine. These disruptions underline the importance of Tyr236 for the integrity of the substrate binding site and the optimal alignment of Arg55, which appears to be necessary for efficient electron transfer.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

PCTH: A novel orally active chelator for the treatment of iron overload disease

Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of this series of chelators, both in cell culture and in animal models. Several PCIH analogs were shown to be effective at inducing iron mobilization and preventing iron uptake from the iron-transport protein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrion and inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orally active and well tolerated by mice at doses ranging from 50 to 100 mg kg(-1) , twice daily (b.d.). A dose-dependent increase in fecal Fe-59 excretion was observed in the PCTH-treated group. This level of iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for the treatment of ss-thalassemia (thal) and Friedreich's Ataxia (FA).

Risk of serious NSAID-related gastrointestinal events during long-term exposure: a systematic review

Objective: Exposure to non-steroidal anti-inflammatory drugs (NSAIDs) is associated wit increased risk of serious gastrointestinal (GI) events compared with non-exposure. We investigated whether that risk is sustained over time. Data sources: Cochrane Controlled Trials Register (to 2002); MEDLINE, EMBASE, Derwent Drug File and Current Contents (1999-2002); manual searching of reviews (1999-2002). Study selection: From 479 search results reviewed and 221 articles retrieved, seven studies of patients exposed to prescription non-selective NSAIDs for more than 6 months and reporting time-dependent serious GI event rates were selected for quantitative data synthesis. These were stratified into two groups by study design. Data extraction: Incidence of GI events and number of patients at specific time points were extracted. Data synthesis: Meta-regression analyses were performed. Change in risk was evaluated by testing whether the slope of the regression line declined over time. Four randomised controlled trials (RCTs) provided evaluable data from five NSAID arms (aspirin, naproxen, two ibuprofen arms, and diclofenac). When the RCT data were combined, a small significant decline in annualised risk was seen: -0.005% (95% Cl, -0.008% to -0.001%) per month. Sensitivity analyses were conducted because there was disparity within the RCT data. The pooled estimate from three cohort studies showed no significant decline in annualised risk over periods up to 2 years: -0.003% (95% Cl, -0.008% to 0.003%) per month. Conclusions: Small decreases in risk over time were observed; these were of negligible clinical importance. For patients who need long-term (> 6 months) treatment, precautionary measures should be considered to reduce the net probability of serious GI events over the anticipated treatment duration. The effect of intermittent versus regular daily therapy on long-term risk needs further investigation.

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at -2 and -278 mV (with a ratio of 1e(-):3e(-)) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were -20 and -254 mV. All redox responses exhibit a pH dependence of approximately -59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry. (c) 2006 Elsevier B.V. All rights reserved.

CULTURA ORGANIZACIONAL: UM ESTUDO SOBRE O SISTEMA DE VALORES DE UMA MULTINACIONAL NO BRASIL

Este trabalho apresenta o tema CULTURA ORGANIZACIONAL: UM ESTUDO SOBRE O SISTEMA DE VALORES DE UMA MULTINACIONAL NO BRASIL . O eixo central da pesquisa é discutir a cultura organizacional global da multinacional sua relação com a cultura organizacional local da subsidiária no Brasil, bem como as relações de compartilhamento. Para isto, utiliza-se como objeto de estudo uma empresa multinacional alemã do segmento de produtos esportivos com subsidiária no Brasil. Com intuito de responder ao objetivo do trabalho foi realizado um estudo de caso único, descritivo explicativo, em uma empresa multinacional alemã instalada na cidade de São Paulo. O caso é composto por 62 sujeitos, 07 gestores e 55 funcionários administrativos que trabalham na subsidiária brasileira. Como resultado obteve-se a idéia de que a cultura organizacional é um fator relevante na sociedade pós-industrial globalizada e é necessário que as empresas multinacionais compreendam a cultura nacional do país onde desejam instalar-se e entendam como administrar as diversidades culturais de cada país.(AU)

Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. The effects of arachidonic acid upon the volume-sensitive Cl- current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole-cell patch-clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10-15 s of application preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time-dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half-maximal inhibitory concentrations (IC50) were 177 +/- 31 and 10 +/- 4 microM for the two phases respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo-oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10-100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume-sensitive Cl- conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Three-factor models versus time series models:quantifying time-dependencies of interactions between stimuli in cell biology and psychobiology for short longitudinal data

Signal integration determines cell fate on the cellular level, affects cognitive processes and affective responses on the behavioural level, and is likely to be involved in psychoneurobiological processes underlying mood disorders. Interactions between stimuli may subjected to time effects. Time-dependencies of interactions between stimuli typically lead to complex cell responses and complex responses on the behavioural level. We show that both three-factor models and time series models can be used to uncover such time-dependencies. However, we argue that for short longitudinal data the three factor modelling approach is more suitable. In order to illustrate both approaches, we re-analysed previously published short longitudinal data sets. We found that in human embryonic kidney 293 cells cells the interaction effect in the regulation of extracellular signal-regulated kinase (ERK) 1 signalling activation by insulin and epidermal growth factor is subjected to a time effect and dramatically decays at peak values of ERK activation. In contrast, we found that the interaction effect induced by hypoxia and tumour necrosis factor-alpha for the transcriptional activity of the human cyclo-oxygenase-2 promoter in HEK293 cells is time invariant at least in the first 12-h time window after stimulation. Furthermore, we applied the three-factor model to previously reported animal studies. In these studies, memory storage was found to be subjected to an interaction effect of the beta-adrenoceptor agonist clenbuterol and certain antagonists acting on the alpha-1-adrenoceptor / glucocorticoid-receptor system. Our model-based analysis suggests that only if the antagonist drug is administer in a critical time window, then the interaction effect is relevant.