[Study of methylation of mitochondrial MT-COI of benzene poisoning].

Objective: To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec µlar biomarkers in patients with occupational chronic benzene poisoning. Methods: 38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups. Results: The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC (P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets (r=0.254、0.280, P<0.05) . Conclusion: The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

Downregulation of GNAS inhibits osteogenesis of bone marrow mesenchymal stem cells and promotes osteoporosis through the Wnt pathway.

OBJECTIVE: This study aims to explore the role of GNAS in accelerating the progression of osteoporosis by inhibiting osteogenesis of BMSCs by the Wnt pathway. PATIENTS AND METHODS: GNAS levels in OP tissues and BMSCs undergoing osteogenesis for different time points were detected. Regulatory effects of GNAS on osteogenesis-related gene expressions, ALP activity, capability of mineralization, and activation of the Wnt pathway in BMSCs were assessed through a series of functional experiments. At last, rescue experiments were performed to further verify the significance of the Wnt pathway during GNAS-mediated osteogenesis development. RESULTS: GNAS was downregulated in OP tissues relative to normal bone tissues. With the prolongation of osteogenesis, GNAS level gradually increased in BMSCs. Knockdown of GNAS downregulated expression levels of ALP and RUNX2, and attenuated ALP activity and capability of mineralization in BMSCs. GNAS was able to activate the Wnt pathway in BMSCs. Notably, overexpression of Wnt3a could reverse the regulatory effects of GNAS on osteogenesis-related gene expressions, ALP activity, and capability of mineralization in BMSCs. CONCLUSIONS: Downregulation of GNAS suppresses osteogenesis of BMSCs through the Wnt pathway, thus aggravating the progression of osteoporosis.

Oxidation of pyridine nucleotides is an early event in the lethality of allyl alcohol.

The involvement of altered pyridine nucleotide concentrations in the cytolethality of allyl alcohol was studied in isolated rat hepatocytes. NAD+, NADH, NADP+, NADPH and viability loss (leakage of lactate dehydrogenase into the medium) were measured in cells incubated with 0.5 mM allyl alcohol with or without the addition of 2 mM dithiothreitol at 30 min. Exposure to allyl alcohol increased NADH levels in the first 15 min of incubation. A sharp drop in NADH and NADPH with an accumulation of NADP+ occurred between 30 and 60 min of incubation with allyl alcohol, indicating an oxidation and interconversion of pyridine nucleotides. Dithiothreitol prevented the oxidation of pyridine nucleotides, but not their reduction or interconversion, and protected against cell killing by allyl alcohol. The results suggest that pyridine nucleotide oxidation might be important for allyl alcohol-induced cytotoxicity; however, a causal relationship between pyridine nucleotide oxidation and cell killing is yet to be demonstrated.

Loss of mitochondrial membrane potential is not essential to hepatocyte killing by allyl alcohol.

Allyl alcohol-induced LDH leakage from isolated rat hepatocytes was preceded by a decrease in rhodmine 123 retention, signifying a loss of mitochondrial membrane potential. Addition of dithiothreitol (DTT) prevented the drop in membrane potential and completely prevented cell killing by allyl alcohol. In contrast, cyclosporin A and trifluoperazine delayed the loss of membrane potential without affecting cytolethality. The results indicate that a drop in mitochondrial membrane potential is not essential for allyl alcohol lethality. The mitochondrial dysfunction produced by allyl alcohol appears to be the consequence of an earlier event in the toxicity that is reversible by DTT.

On the dynamics of bursting systems.

The dynamics of three-variable models of bursting are studied. It is shown that under certain conditions, the dynamics on the attractor can be essentially reduced to two dimensions. The salient dynamics on the attractor can thus be completely described by the return map of a section which is a logistic interval map. Two specific bursting models from the literature are shown to fit in the general framework which is developed. Bifurcation of the full system for one case in investigated and the dynamical behavior on the attractor is shown to depend on the position of a certain nullcline.

Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity.

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.

On the reactions of lignin peroxidase compound III (isozyme H8).

Compound III (oxyperoxidase) of lignin peroxidase isozyme H8 (pI = 3.5) is formed by either reduction of native ferric enzyme (to ferrous) followed by the reaction with dioxygen or by the addition of excess hydrogen peroxide to resting enzyme. When prepared from the ferrous enzyme, Compound III is stable for days. When formed from excess hydrogen peroxide, the enzyme is rapidly inactivated. However, if the hydrogen peroxide is removed by gel filtration, the resulting Compound III exhibits the same stability as when prepared from ferrous enzyme. Compound III of lignin peroxidase is also relatively unreactive to reducing substrates. Addition of veratryl alcohol to Compound III does not result in any reaction. However, when only 1 equivalent of hydrogen peroxide is added to Compound III in the presence of veratryl alcohol, Compound III is converted to resting enzyme and veratraldehyde formation is detected spectroscopically.

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies.

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium.

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.