Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.     

Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of the metal clusters in the DeltanifB and DeltanifH mofe proteins of nitrogenase from Azotobacter vinelandii

Deletion of nifB results in the formation of a variant nitrogenase MoFe protein (DeltanifB MoFe protein) that appears to contain two normal [8Fe-7S] P clusters. This protein can be reactivated to form the holo MoFe protein upon addition of isolated FeMo cofactor. In contrast, deletion of nifH results in a variant protein (DeltanifH MoFe protein) that appears to contain FeS clusters different from the normal P cluster, presumably representing precursors of the normal P cluster. The DeltanifH MoFe protein is not reconstituted to the holo MoFe protein with isolated FeMo cofactor. The EPR and EXAFS spectroscopic properties of FeS clusters in the DeltanifH MoFe protein clearly differ from those of the normal P cluster found in the DeltanifB MoFe protein and suggest the presence of [4Fe-4S]-like clusters. To further characterize the metal cluster structures in the DeltanifH MoFe protein, a variable-temperature, variable-field magnetic circular dichroism (VTVH-MCD) spectroscopic study has been undertaken on both the DeltanifB MoFe protein and the DeltanifH MoFe protein in both the dithionite-reduced and oxidized states. This study clearly shows that each half of the dithionite-reduced DeltanifH MoFe protein contains a [4Fe-4S]+ cluster paired with a diamagnetic [4Fe-4S]-like cluster. Upon oxidation, the VTVH-MCD spectrum of the DeltanifH MoFe protein reveals a paramagnetic, albeit EPR-silent system, suggesting an integer spin state. These results suggest that the DeltanifH MoFe protein contains a pair of neighboring, unusual [4Fe-4S]-like clusters, which are paramagnetic in their oxidized state.     

Down-regulation of ABCB1 transporter by atorvastatin in a human hepatoma cell line and in human peripheral blood mononuclear cells

PURPOSE: The effect of atorvastatin, an HMG-CoA reductase inhibitor, on expression and activity of the drug transporter ABCB1 in HepG2 cells and peripheral blood mononuclear cells (PBMCs) was examined. METHODS: Localization and expression of ABCB1 in hepatocytes was examined by indirect immunofluorescence. Expression of ABCB1 mRNA and ABCB1 activity were examined in atorvastatin-treated and control cells and PBMCs using real-time PCR and Rhodamine 123 efflux assay. RESULTS: Immunohistochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi. Atorvastatin at 10 and 20 microM up-regulated ABCB1 expression resulting in a significant 1.4-fold increase of the protein levels. Treatment of HepG2 cells with 20 microM atorvastatin caused a 60% reduction on mRNA expression (p<0.05) and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p<0.01) by flow cytometry. Correlation was found between ABCB1 mRNA levels and creatine kinase (r=0.30; p=0.014) and total cholesterol (r=-0.31; p=0.010). CONCLUSIONS. Atorvastatin leads to decreased ABCB1 function and modulates ABCB1 synthesis in HepG2 cells and in PBMCs. ABCB1 plays a role in cellular protection as well as in secretion and/or disposition, therefore, inhibition of ABCB1 synthesis may increase the atorvastatin efficacy, leading to a more pronounced reduction of plasma cholesterol.     

Low temperature-acclimation impairs cellular migration in the adult cerebral cortex of the tropical lizard, Tropidurus hispidus (Spix, 1825) (Squamata: Tropiduridae)

The effects of different temperature-acclimations on cellular proliferation and migration were studied in the cerebral cortex of the tropical lizard, Tropidurus hispidus. Lizards were divided in two groups: warm-acclimated lizards (WALs), maintained at the temperature and photoperiod conditions of their natural habitat (mean temperature 26°C; 12:12 light:dark) and the cold-acclimated lizards (CALs), maintained at the same cycle of illumination and a mean temperature of 16°C. Animals were injected with the proliferative marker 5-bromodeoxyuridine (BrdU) and euthanized fifteen or thirty days later for the immunostaining. There was no difference in the number of BrdU-positive nuclei between the experimental groups in any of the cortical layers. In CALs, the positive nuclei were found mostly close to the ependyma, whereas in WALs many positive nuclei were also found in the plexiform and cellular layers of the cortex. In CALs, BrdU-positive nuclei appeared grouped (of 2-3 nuclei), a characteristic not seen in the other group. These data suggest that temperature affects the migrating capability of the newly generated neurons in the lizard cortex, but appears not to interfere with its generation.     

Kinetic characterization of gyroxin,a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.     

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.     

An outpatient-based clinical program for diabetes prevention: an update

ObjectiveTo update outcomes of the Diet-ExerciseActivity-Lifestyle (DEAL) program, a clinic-based diabetes prevention intervention.MethodsChanges in weight, fasting blood glucose, and 2-hour glucose after a 75-g oral glucose tolerance test were evaluated in patients who enrolled in the DEAL program between January 2007 and August 2009.ResultsThe 221 qualified participants had a mean age of 62 years, weight of 87.4 kg, body mass index of 31.2 kg/m², fasting glucose level of 109 mg/dL, and 2-hour glucose value of 138 mg/dL. Among the program participants, 67% were women and 88% were white; 56% had isolated impaired fasting glucose, 5% had impaired glucose tolerance only, and 39% had both. The 6-month follow-up medical appointment was kept by 72% of program participants, but only 56% attended the 12-month visit. By 6 months after baseline, 59% had significantly lower fasting glucose concentrations, 59% had improvement in 2-hour glucose levels, and 61% had weight loss. Nearly 40%, however, were nonresponders and had increased fasting glucose, 2-hour glucose, and weight by 6 months. By the 12-month visit, significant declines in fasting glucose (P < .001), 2-hour glucose (P < .001), and weight (P = .008) occurred in comparison with baseline values; however, no significant changes occurred in these measures between the 6and 12-month visits (P > .30 for all).ConclusionMost DEAL participants showed improvement in glucose levels and weight, but some patients exhibited worsening glucose intolerance. Factors underlying nonresponse need to be identified. Ongoing experience and analysis should help revise the DEAL program so that outcomes for all participating patients will improve. (Endocr Pract. 2012;18:200-208)     

Prevalence of esophageal atresia among 18 international birth defects surveillance programs

BACKGROUND: The prevalence of esophageal atresia (EA) has been shown to vary across different geographical settings. Investigation of geographical differences may provide an insight into the underlying etiology of EA. METHODS: The study population comprised infants diagnosed with EA during 1998 to 2007 from 18 of the 46 birth defects surveillance programs, members of the International Clearinghouse for Birth Defects Surveillance and Research. Total prevalence per 10,000 births for EA was defined as the total number of cases in live births, stillbirths, and elective termination of pregnancy for fetal anomaly (ETOPFA) divided by the total number of all births in the population. RESULTS: Among the participating programs, a total of 2943 cases of EA were diagnosed with an average prevalence of 2.44 (95% confidence interval [CI], 2.35–2.53) per 10,000 births, ranging between 1.77 and 3.68 per 10,000 births. Of all infants diagnosed with EA, 2761 (93.8%) were live births, 82 (2.8%) stillbirths, 89 (3.0%) ETOPFA, and 11 (0.4%) had unknown outcomes. The majority of cases (2020, 68.6%), had a reported EA with fistula, 749 (25.5%) were without fistula, and 174 (5.9%) were registered with an unspecified code. CONCLUSIONS: On average, EA affected 1 in 4099 births (95% CI, 1 in 3954–4251 births) with prevalence varying across different geographical settings, but relatively consistent over time and comparable between surveillance programs. Findings suggest that differences in the prevalence observed among programs are likely to be attributable to variability in population ethnic compositions or issues in reporting or registration procedures of EA, rather than a real risk occurrence difference. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.     

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.     

New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.