Gamma-Carboxylation and Fragmentation of Osteocalcin in Human Serum Defined by Mass Spectrometry

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0–3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.Osteocalcin (Oc)¹ is a member of the family of vitamin K-dependent gamma (γ)-carboxylated proteins. The formation of γ-carboxyglutamic acid (Gla) occurs via the carboxylation of three specific glutamic acid residues in the mid-molecular region of Oc (E17, E21, and E24) and results in the binding of Oc to hydroxyapatite in bone (1). In circulation Oc is a highly specific bone marker that has been used for assessing relative degrees of bone turnover in clinical studies (2). Based on immunological methods, a general notion has been that the major forms of circulating Oc are the intact molecule and a large N-terminal-mid molecule fragment encompassing residues 1–43, thought to be the result of trypsin-like activity in serum or poor sample handling (3). However, the precise sequence has never been clearly defined and considerable inconsistency is evident when comparing values from different laboratories or commercial kits because of differences in antibody specificity.Carboxylated variants of Oc are also found both in human bone and in circulation. Initial observational studies that reported an association between poor vitamin K status and bone loss attributed the finding to an increased proportion of Oc in circulation that was not carboxylated, reflecting a nonfunctional protein in bone (4–6). However, assays for total Oc are indifferent to carboxylation status, and methods used to measure the carboxylation state of circulating Oc do not distinguish among the fully, partially, or uncarboxylated forms (7, 8). Therefore, even though the amount of undercarboxylated Oc relative to the total in circulation (%ucOC) is a biomarker of vitamin K status in bone, there is no consensus on the precise amount in circulation or how many of the three potential Gla residues are carboxylated.Recently, mouse models have indicated that circulating Oc also serves as an endocrine hormone with a positive role in glucose metabolism (9). Paradoxically, the active form is un- or undercarboxylated, whereas the carboxylated form is inactive (10). A growing number of human studies have examined associations between total Oc and baseline or changing levels of fasting glucose, insulin, or HOMA-IR (11). However, few have directly measured the putative active form of the protein or taken into account that the carboxylation of Oc is very sensitive to daily fluctuations in intakes of vitamin K provided in such food sources as plant oils such as olive, canola and soybean, and green vegetables, such as broccoli, spinach, and kale (12, 13).Based on results provided by mass spectrometric immunoassay (MSIA), we herein report new qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual blood plasma and serum samples. We further present molecular details on the responses of specific carboxylated forms and fragments of Oc in plasma of free-living older adults who received different amounts of vitamin K under controlled conditions. Such determinations of Oc γ-carboxylation in individual serum samples will ultimately be necessary to translate the functional significance of fluctuating levels of Oc in human health and disease.     

Coupling of M3 acetylcholine receptor to Gq16 activates a natriuretic peptide receptor guanylyl cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M(3)AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Galpha(i/o) subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Galpha(q16), whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Galpha(q15/16) stimulated the NPR-GC. Coupling of alpha(q16) to M(3)AChR is supported by MAS decreased [(3)H]QNB binding, being abolished after M(3)AChR-4-DAMP-alkylation. Anti-i(3)M(3)AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i(3)M(3)AChR (M(3)P) was more potent than MAS increasing GTPgamma [(35)S] and decreasing the [(3)H]QNB activities. Coupling between NPR-GC and Galpha(q16) was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Galpha(q16), also showing an immunoreactive heterotrimeric-G-beta-subunit. These data support the existence of a novel transducing cascade, involving Galpha(q16)beta gamma coupling M(3)AChR to NPR-GC.     

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii

Mycopathologia - Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix...     

Early Interaction of Alternaria infectoria Conidia with Macrophages

Mycopathologia - Fungi of the genus Alternaria are ubiquitous indoor and outdoor airborne agents, and individuals are daily exposed to their spores. Although its importance in human infections and,...     

Impact of Income Inequality and Other Social Determinants on Suicide Rate in Brazil

Studies about suicide worldwide have mainly focused on individual-level psychiatric risk factors. In Brazil, suicide is an important public health problem. Brazil has evidenced important socioeconomic changes over the last decades, leading to decreasing income inequality. However, the impact of income inequality on suicide rate has never been studied in the country.

Purpose

To analyze whether income inequality and other social determinants are associated with suicide rate in Brazil.

Method

This study used panel data from all 5,507 Brazilian municipalities from 2000 to 2011. Suicide rates were calculated by sex and standardized by age for each municipality and year. The independent variables of the regression model included the Gini Index, per capita income, percentage of individuals with up to eight years of education, urbanization, average number of residents per household, percentage of divorced people, of Catholics, Pentecostals, and Evangelicals. A multivariable negative binomial regression for panel data with fixed-effects specification was performed.

Results

The Gini index was positively associated with suicide rates; the rate ratio (RR) was 1.055 (95% CI: 1.011–1.101). Of the other social determinants, income had a significant negative association with suicide rates (RR: 0.968, 95% CI: 0.948–0.988), whereas a low-level education had a positive association (RR: 1.015, 95% CI: 1.010–1.021).

Conclusions

Income inequality represents a community-level risk factor for suicide rates in Brazil. The decrease in income inequality, increase in income per capita, and decrease in the percentage of individuals who did not complete basic studies may have counteracted the increase in suicides in the last decade. Other changes, such as the decrease in the mean residents per household, may have contributed to their increase. Therefore, the implementation of social policies that may improve the population’s socioeconomic conditions and reduce income inequality in Brazil, and in other low and middle-income countries, can help to reduce suicide rates.     

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.     

DNA damage-induced cell death： lessons from the central nervous system

DNA damage can, but does not always, induce cell death. While several pathways linking DNA damage signals to mitochondria-dependent and -independent death machineries have been elucidated, the connectivity of these pathways is subject to regulation by multiple other factors that are not well understood. We have proposed two conceptual models to explain the delayed and variable cell death response to DNA damage： integrative surveillance versus autonomous pathways. In this review, we discuss how these two models may explain the in vivo regulation of cell death induced by ionizing radiation （IR） in the developing central nervous system, where the death response is regulated by radiation dose, cell cycle status and neuronal development.     

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.