Evaluation of Relationship between Occurrence of Liver Cancer and Methylation of Fragile Histidine Triad (FHIT) and P16 Genes.

BACKGROUND We examined the level of fragile histidine triad (FHIT) and p16 gene methylation in patients with hepatocellular carcinoma and explored the relationship to liver cancer. MATERIAL AND METHODS There were 56 patients with primary liver cancer who were admitted to the hospital from July 2015 to October 2017 included in the liver cancer group, and 24 non-hepatoma patients (hepatitis A/hepatitis B/hepatitis C, liver cirrhosis, liver fibrosis, and fatty liver, alcoholic liver identified as a control group. Fasting venous blood samples were collected from the 2 groups. Methylation-specific PCR (MSP) was used to detect the methylation of FHIT and p16 genes in the 2 groups. The risk factors for lung cancer were analyzed by logistic regression. In addition, the effects of FHIT and p16 gene methylation on the diagnostic accuracy of liver cancer were calculated. RESULTS The incidence of FHIT and p16 methylation in serum from the liver cancer group was 51.8% and 67.9%, respectively. The incidence of FHIT and p16 methylation in the non-hepatoma group was 16.7% and 25.0%. There was a statistical statistically correlated with gender, and the methylation of FHIT and p16 genes (P<0.05). From logistic regression analysis results, methylation of p16 and FHIT genes was an independent risk factor for hepatocellular carcinoma with odds ratio (OR) values of 10.550 (2.313~48.116) and 8.239 (2.386~28.455), respectively. CONCLUSIONS The methylation of p16 and FHIT genes was an independent risk factor for hepatocellular carcinoma.

Enhanced Activities of Blood Thiamine Diphosphatase and Monophosphatase in Alzheimer's Disease.

BACKGROUND: Thiamine metabolites and activities of thiamine-dependent enzymes are impaired in Alzheimer's disease (AD). OBJECTIVE: To clarify the mechanism for the reduction of thiamine diphosphate (TDP), an active form of thiamine and critical coenzyme of glucose metabolism, in AD. METHODS: Forty-five AD patients clinically diagnosed and 38 age- and gender-matched control subjects without dementia were voluntarily recruited. The contents of blood TDP, thiamine monophosphate (TMP), and thiamine, as well as the activities of thiamine diphosphatase (TDPase), thiamine monophosphatase (TMPase), and thiamine pyrophosphokinase (TPK), were assayed by high performance liquid chromatography. RESULTS: Blood TDP contents of AD patients were significantly lower than those in control subjects (79.03 ± 23.24 vs. 127.60 ± 22.65 nmol/L, P<0.0001). Activities of TDPase and TMPase were significantly enhanced in AD patients than those in control subjects (TDPase: 1.24 ± 0.08 vs. 1.00 ± 0.04, P < 0.05; TMPase: 1.22 ± 0.04 vs. 1.00 ± 0.06, P < 0.01). TPK activity remained unchanged in AD as compared with that in control (0.93 ± 0.04 vs. 1.00 ± 0.04, P > 0.05). Blood TDP levels correlated negatively with TDPase activities (r = -0.2576, P = 0.0187) and positively with TPK activities (r = 0.2426, P = 0.0271) in all participants. CONCLUSION: Enhanced TDPase and TMPase activities may contribute to the reduction of TDP level in AD patients. The results imply that an imbalance of phosphorylation-dephosphorylation related to thiamine and glucose metabolism may be a potential target for AD prevention and therapy.

Alterations of ectonucleotidases and acetylcholinesterase activities in lymphocytes of Down syndrome subjects: relation with inflammatory parameters.

BACKGROUND: Subjects with Down syndrome (DS) have an increased susceptibility to infections and autoimmune disorders. ATP, adenosine, and acetylcholine contribute to the immune response regulation, and NTPDase, adenosine deaminase (ADA) and acetylcholinesterase (AChE) are important enzymes in the control of the extracellular levels of these molecules. We evaluated the activities of these enzymes and the cytokine levels in samples of DS individuals. METHODS: The population consisted of 23 subjects with DS and 23 healthy subjects. Twelve milliliters of blood was obtained from each subject and used for lymphocyte and serum preparation. Lymphocytes were separated on Ficoll density gradients. After isolation, NTPDase and AChE activities were determined. RESULTS: The NTPDase activity using ADP as substrate was increased in lymphocytes of DS patients compared to control (P<0.05); however, no alterations were observed in the ATP hydrolysis. An increase was observed in the AChE activity in lymphocytes and in ADA activity in serum of DS patients when compared to healthy subjects (P<0.05). In DS subjects, an increase in the levels of IL-1ß, IL-6, TNF-α and IFN-γ and a decrease in the IL-10 levels were also observed (P<0.05). CONCLUSIONS: Alterations in the NTPDase, ADA and AChE activities as well changes in the cytokine levels may contribute to immunological alterations observed in DS.

[Methylation status of FHIT gene in plasma and expression of FHIT gene in cancer tissue of cervical cancer patients].

OBJECTIVE: To explore the methylation status of 5' CpG island of fragile histidine triad (FHIT) gene in plasma and the expression of FHIT protein in cancer tissue of cervical cancer patients. METHODS: Methylation-specific PCR (MS-PCR) was employed to examine methylation of FHIT gene in 151 plasma samples before treatment. The immunohistochemistry was used to the expression of FHIT protein in cancer tissues. RESULTS: CpG island methylation of FHIT was detected in 31.13% of plasma samples. The expression of FHIT protein was decreased or discarded in 59.60% of cervical cancer tissues. Among them 47.78% was included in methylation positive samples. CONCLUSION: CpG island methylation of FHIT gene in plasma plays an important role on cervical cancer, which results in decreased expression of FHIT protein. It can be used to diagnose and evaluate the effect of treatment to cervical cancers.

[Methylation status of the 5' CpG islands in FHIT gene in the plasma and tissues of cervical cancer patients].

To evaluate the methylation status of the 5' CpG islands in FHIT gene using plasma and tissue samples from cervical cancer patients and find a novel marker for non-invasive diagnosis of cervical cancer, methylation-specific PCR (MSP) was employed to examine CpG island methylation in FHIT gene in 151 pretreatment plasma samples and 30 tumor tissue samples obtained from cervical cancer patients. MSP product was cloned and sequenced directly. CpG island methylation of FHIT was detected in 31.13% of the plasma samples, and in 53.33% of the tissue samples. The total concordant rate of methylation status between plasma and tissue samples in FHIT gene was 80.00%. We found a strong positive correlation between FHIT methylation in the plasma and the clinical stage and histological grade of the tumor. The data showed that CpG island methylation of the FHIT gene is prevalent in the plasma and tissue samples from cervical cancer patients. FHIT detection may be used as a non-invasive marker for diagnosis of cervical cancer and prognostic treatment evaluation.

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein.

We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.

[Liquid chromatographic determination of free thiamine and its esters in whole blood]. / Determination de la thiamine libre et de ses esters dans le sang total par chromatographie liquide.

INTRODUCTION: To appreciate vitamin B1 status in normal and pathophysiological states in local ivorian populations, a high-performance liquid chromatographic method for the simultaneous determination of thiamine and its phosphate esters: thiamine monophosphate and thiamine diphsophate or cocarboxylase in whole blood has been developed. METHODS: The method involves extraction with diethylether, followed by pre-column alkaline derivatization by bromide cyanogen. The extract was analysed by isocratic reversed-phase isocratic high performance liquid chromatography with fluorescent detection. System suitability tests were applied to assess the method's continuing suitability for use. The main tests of method validation: linearity, precision, accuracy and sensibility were applied to the analytical procedure. RESULTS: Thiamine and its esters were eluted within 10 minutes with a good resolution. System suitability tests showed that the chromatographic system was suitable for continuing use. Results of validation tests show the reliability of the method : linearity domain, satisfying precision and accuracy, detection limits between 0.067 pg/l et 0.92 pg/l (0.160 fmol/l et 1.822 fmol/l). CONCLUSION: The proposed method is suitable for determination of thiamine and its phosphate esters in whole blood.

Kinetics of the nucleoside triphosphate hydrolase of Toxoplasma gondii in mice with acute and chronic toxoplasmosis.

The kinetics of the nucleoside triphosphate hydrolase (NTPase) of Toxoplasma gondii was examined using an avidin-biotin sandwich-ELISA (ABS-ELISA) based on an anti-NTPase monoclonal antibody, 6C6. The RH and ME49 strains of the parasite were used to produce acute and chronic infections in mice, respectively. In the acute model, detectable serum concentrations of NTPase were observed from day 1 post-infection and gradually increased until the death of the mice. They were associated with parasitaemia (as estimated by bioassay). No anti-T. gondii antibody could be detected at any time. In the chronic model, in which 20 T. gondii ME49 cysts were given to each mouse per os, the NTPase concentration generally increased from day 3, peaked between days 7 and 14 and then declined. However, one of the four mice used still had a high serum concentration of NTPase on day 35. Again, detectable NTPase concentrations occurred when the mice had parasitaemias. Antibody to T. gondii was detected from day 7 (IgM) or 10 (IgG) and brain cysts were observed from day 14. Since detectable serum concentrations of NTPase appear to be associated with parasitaemia in both acute and chronic toxoplasmosis, the ABS-ELISA of the enzyme may make a useful diagnostic tool.

Human diadenosine triphosphate hydrolase: preliminary characterisation and comparison with the Fhit protein, a human tumour suppressor.

Human platelets diadenosine triphosphatase was characterised and compared with the Fhit protein, a human tumour suppressor with diadenosine triphosphatase activity. Both enzymes exhibit similar Km, are similarly activated by Mg2+, Ca2+ and Mn2+, and inhibited by Zn2+ and suramin. However, they are differentially inhibited by Fhit antibodies and exhibit differences in gel-filtration behaviour.

dNTP modified at triphosphate residues: substrate properties towards DNA polymerases and stability in human serum.

Substrate and terminating substrate properties of dNTP with phosphate groups replaced by phosphonates at alpha-, gamma-, beta, gamma-, and alpha, beta, gamma-positions towards different human DNA polymerases and retroviral reverse transcriptases are reviewed. Substitution of the phosphate group by the phosphonate at any of the three phosphate positions of dNTP increased their stability towards dephosphorylating enzymes of human blood. In some cases hydrophobicity of these compounds was markedly enhanced.

Diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets.

An asymmetrically-cleaving diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase (Ap4A-->ATP+AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap4A as an intra- and extracellular signalling molecule. Ap4A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap4A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M(r) 19,200. The erythrocyte and platelet enzymes had a Km for Ap4A of 0.70 +/- 0.05 microM (n = 3) while the Km for the leukocyte enzyme was 1.50 +/- 0.20 microM (n = 3). All three enzymes showed substrate inhibition above 10 microM Ap4A. The specific activity of the enzyme in erythrocytes was 0.067 U/10(6) cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap4A hydrolase activity. The last observation is of particular interest given the reported absence of Ap4A from enucleated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, kinetic properties and primary structure of bovine erythrocyte acylphosphatase.

An erythrocyte isoenzyme of acylphosphatase was purified from bovine red cells. The protein was characterized as regards the kinetic parameters and amino acid sequence. A simple and rapid sequencing strategy, based on a few experiments, was used for reconstructing the primary structure of the enzyme, since the purification procedure gave a very low yield. The length of the polypeptide chain is 100 residues. Comparison with the analogous human isoenzyme indicates that the primary structure is about 90% conserved. The presence of two additional residues at the acetylated N-terminus confirms the hypervariability for this region found in other acylphosphatases.