Hepatitis B Virus Core Promoter Double Mutations (A1762T, G1764A) Are Associated with Lower Levels of Serum Dihydrolipoyl Dehydrogenase.

OBJECTIVES: The aim of this study was to identify serum proteins with differential concentrations between hepatocellular carcinoma (HCC) patients and HBsAg asymptomatic carriers among individuals infected with hepatitis B virus (HBV) with basal core promoter (BCP) double mutations (A1762T, G1764A). METHODS: iTRAQ and liquid chromatography-tandem mass spectrometry were used to identify differentially expressed protein, and an ELISA test was used for the validation test. RESULTS: The total number of proteins identified was 1,125, of which 239 showed statistically significant differences in their expression. The relative concentrations of serum dihydrolipoyl dehydrogenase (DLD), which showed the most significant correlation with liver diseases and infection, were significantly lower in HCC patients than asymptomatic HBsAg carriers and individuals negative for HBsAg. However, only the difference between HCC patients with BCP double mutations and HBsAg-negative individuals could be confirmed by ELISA. Meanwhile, we found that the concentrations of serum DLD in those infected with HBV with BCP double mutations were significantly lower than in individuals with the wild-type BCP. However, the difference in the concentrations of serum DLD between individuals with wild-type BCP and those negative for HBsAg was not significant. CONCLUSIONS: HBV with BCP double mutations are associated with lower concentrations of serum DLD.

Nongradient blue native gel analysis of serum proteins and in-gel detection of serum esterase activities.

The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either α-naphthylacetate or ß-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.

Radiofrequency ablation as local therapy for early breast carcinomas.

PURPOSE: To evaluate the safety and efficacy of radiofrequency ablation (RFA) as a local therapy for early breast carcinomas, we performed a phase I/II study at our institution. PATIENTS AND METHODS: Fifty patients with core-needle biopsy-proven breast carcinoma that was ≤ 3 cm in diameter on ultrasonography were enrolled in this study. Under ultrasound (US) guidance, the tumor and surrounding breast tissue were ablated with a saline-cooled RF electrode followed by immediate surgical resection. Resected specimens were examined by hematoxylin and eosin (H&E) staining and nicotinamide adenine dinucleotide (NADH) diaphorase staining to assess tumor viability. RESULTS: Forty-nine patients completed the treatment. The mean tumor size was 1.70 cm. The mean ablation time was 8.7 min using a mean power of 48.5 W. Of the 49 treated patients, complete ablation was recognized in 30 patients (61%) by H&E staining and/or NADH diaphorase staining. The NADH viability staining was available for 38 patients, and in 29 (76.3%), there was no evidence of viable malignant cells. Of the 29 treated patients with breast carcinomas ≤ 2 cm in diameter examined by pathological examination, complete ablation was achieved in 24 patients (83%). Of the 26 treated patients with breast carcinomas without an extended intraductal component (EIC) according to pathological examination, complete ablation was determined in 22 patients (85%). RFA-related adverse events were observed in five cases: two with skin burn and three with muscle burns. CONCLUSION: RF ablation is a safe and promising minimally invasive treatment for small breast carcinomas with pathological tumor size ≤ 2 cm in diameter and without EIC.

Pilot study of radiofrequency ablation therapy without surgical excision for T1 breast cancer: evaluation with MRI and vacuum-assisted core needle biopsy and safety management.

BACKGROUND: There is increasing demand for minimally invasive treatments for small breast cancer mainly because of the desire for better cosmetic results. Although radiofrequency ablation (RFA) is an attractive approach as a local control method for small breast cancer, the problems of histological effectiveness and safety management remain. METHODS: A total of 29 patients including one patient with bilateral breast cancer were enrolled in this study. The mean tumor size of 30 breasts was 12.8 mm (range 5-19 mm). Under general anesthesia, RFA was performed with a Cool-tip RF system (Valleylab, Boulder, CO, USA) after sentinel lymph node biopsy. Postoperative evaluation with magnetic resonance imaging (MRI) and vacuum-assisted core needle biopsy was done 3-4 weeks after RFA before radiotherapy. Ablated tumors were evaluated with hematoxylin-eosin (H&E) and nicotinamide adenine dinucleotide (NADH)-diaphorase staining. If needed, adjuvant chemo and/or endocrine therapy was performed. RESULTS: All patients except one completed one session of RFA. The mean temperature near the center of the tumors was 89.6°C (range 78-100°C). Postoperative MRI showed the ablated zone clearly in all patients. MRI revealed no hypervascularity of the tumors in the ablated zone. Evaluation with H&E staining of the tumors showed remarkable degenerative changes in only three patients. NADH-diaphorase staining showed no viable tumor tissue in 24 patients out of 26 examined. Three patients received small diameter grade 3 skin burns, two on the outside of the thigh from the grounding pad and one on the breast skin. One patient had a breast lesion like a chronic granulomatous mastitis resulting from overreaction of the ablated zone. CONCLUSIONS: RFA therapy appeared relevant and applicable for patients with small breast cancer. Because small skin burns were observed as adverse events, close attention should be paid in the course of the RFA procedure.

Antioxidant adaptive response of human mononuclear cells to UV-B: effect of lipoic acid.

Supplementation of human mononuclear cells with 3 and 6 mM of lipoic acid produces an inhibition of the antioxidant adaptive response triggered by treatment with UV-B light (0.30 W/m2 for 15 min). Supplementation with 1.5 mM of lipoic acid gives no conclusive results. The adaptive response is characterized by an increase in the activities of superoxide dismutase, catalase, glutathione peroxidase and DT-diaphorase. Catalase (5.5 +/- 0.6 pmol/mg prot) increases its activity by up to 22 +/- 3 pmol/mg prot, after irradiation with UV-B. Supplementation with 3 and 6 mM of lipoic acid completely inhibits the adaptive response. The activities of the membrane-bound mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase do not increase after UV-B exposure. Moreover, their activities are found to decrease and the addition of lipoic acid does not prevent this effect. The inhibition of the antioxidant response by lipoic acid in human cells appears as indirect evidence of the existence of oxidative stress in the development of this response. As lipoic acid behaves as an effective antioxidant, it seems that its action decreases the intracellular oxidative signals necessary to develop the adaptive response in human mononuclear cells.

The NADPH oxidase diaphorase activity in permeabilized human neutrophils and granulocytic like PLB-985 cells.

The phagocyte NADPH oxidase is a multicomponent transport chain that generates superoxide, a precursor of microbicidal oxidants, important for host defense. This transport chain is contained mainly in the large membrane subunit of the oxidase (gp91phox), and transfers electrons from cytosolic NADPH, through FAD binding and heme centers, to molecular oxygen (Babior, 1999; Fujii and Kakinuma, 1991; Rotrosen et al., 1992; Segal and Abo, 1993). Cross et al. have recently described a novel NADPH oxidase diaphorase activity present in the membrane fraction of activated neutrophils, using a cell free model (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor 4-iodonitrotetrazolium violet (INT) and is attributed to the reduction of the flavin center of the flavocytochrome (Cross et al., 1994; Li and Guillory, 1997). In the present study we establish a system for detecting diaphorase activity in intact cells. Neutrophils and PLB-985 cells, that were differentiated using 1.25% dimethyl sulfoxide (DMSO) to granulocyte phenotype, were permeabilized by electroporation, and diaphorase activity was determined using INT. Neutrophils and differentiated PLB-985 cells stimulated by PMA or GTP gamma S showed a diaphorase activity that was not present in unstimulated differentiated cells. The diaphorase activity could not be detected in undifferentiated cells and was developed during differentiation. The pattern of diaphorase activity in stimulated parent differentiated PLB cells was similar to that observed in stimulated human neutrophils. The permeabilized-INT cell system offers a unique tool for the evaluation of NADPH oxidase diaphorase activity, in whole cells.

Lipoamide dehydrogenase activity in lymphocytes.

To assess the suitability of lymphocytes for patient diagnosis and carrier detection of lipoamide dehydrogenase deficiency, the activity of lipoamide dehydrogenase was determined in lymphocytes of six patients, seven obligate heterozygotes and 32 healthy controls. In healthy controls, lipoamide dehydrogenase activity was 80.7 +/- 23.6 nmol/min/mg protein, in obligate heterozygotes it was 36.0 +/- 12.1 nmol/min/mg protein and in the patients it was 13.3 +/- 5.1 nmol/min/mg protein. For the purpose of standardization, the results were also calculated as lipoamide dehydrogenase/citrate synthase activity ratio. The activity of lipoamide dehydrogenase and the lipoamide dehydrogenase/citrate synthase ratio differed significantly between the three groups (P < 0.005). We conclude that lymphocytes are suitable for the diagnosis of lipoamide dehydrogenase deficiency and for carrier detection.

Genetic polymorphism of erythrocyte diaphorase in red deer, Cervus elaphus L.

NADH diaphorase polymorphism was identified in red deer erythrocyte lysates using starch gel electrophoresis and activity staining. The inheritance of the polymorphism was consistent with predictions of autosomal codominant inheritance of two alleles DIA1F and DIAS. In New Zealand's four main feral red deer populations (n = 188) the DIA1F allele frequency ranged from 0.491 to 0.985. A sample of North American wapiti (n = 42) was monomorphic for the DIA1F allele.

[NAD- and NADP-diaphorase activity in the peripheral blood lymphocytes of patients with the B-cell variant of chronic lympholeukemia]. / Aktivnost' NAD- i NADP-diaforaz v limfotsitakh perifericheskoi krovi bol'nykh B-kletochnym variantom khronicheskogo limfoleikoza.

The authors studied the cytotoxic function, activity of NAD- and NADP-diaphorases in the peripheral blood lymphocytes in 57 patients with B-cellular variant of chronic lympholeukoses and found a significant reduction of the natural killer activity of lymphocytes, increased activity of NADP-diaphorase. Reduction of natural killer activity in patients with B-cell variant of chronic lympholeukoses did not depend on the activity of membrane diaphorases in peripheral blood lymphocytes.

NADH diaphorase polymorphism in goat erythrocytes.

This paper describes for the first time polymorphism of the erythrocyte diaphorase in goats. Three diaphorase 1 phenotypes were observed in the red cells of goats. Breeding data indicated that polymorphism was controlled by two autosomal codominant alleles, DiaF and DiaS, the frequencies of which were determined in 14 different Spanish breeds of goat.

Hemotypes in Spanish sheep breeds.

The structure and the genetic diversity of the Churra, Lacha and Manchega sheep breeds have been analysed using hemotypes observed in eight loci. The three breeds are different in their hemotypes in terms both of quantity and quality. The proportions of unique hemotypes in Churra (60%), Lacha (61%) and Manchega (67%) revealed a high level of individual diversity within each breed. Racial genetic diversity follows the descending order of: Manchega-Lacha-Churra. The value of N:H (number of animals: total hemotypes) in the multi-racial population was 3.05.

Study of human red cell NADH diaphorase (Dia1) in the Italian population.

A total of about 4,500 individuals from Northern, Central and Southern Italy have been analyzed for red cell NADH diaphorase. The results show that the Italians differ significantly (p less than 0.005) from the other examined populations of European origin by showing a higher frequency of the Dia2 allele (6.4%) and a lower frequency of other Dia variants (0.6%).

Red cell NADH diaphorase variants in Japanese.

Human red cell NADH diaphorase isozyme patterns were examined in 5,046 healthy adult Japanese by starch gel electrophoresis. Twenty had Dia 2-1 and 3 had Dia 4-1 phenotypes. The incidence of Dia variants in patients with mental retardation, cerebral palsy, epilepsy and Down's syndrome was also examined and compared with that of healthy people. It was noticed that thin-layer isoelectric focusing on polyacrylamide gel was very useful for discriminating variant bands from 'aging bands'.

An enzymatic method for the analysis of formate in human plasma.

An enzymatic method for the determination of plasma formate concentration is described. Formate dehydrogenase is used to reduce NAD+ to NADH in the presence of formate. The resulting NADH then reduces the dye resazurin to resorufin, a reaction catalyzed by the endogenous diaphorase of the plasma. The generated resorufin is then measured fluorimetrically by exciting it at 565 nm and quantitating the emitted light at 590 nm. The method uses the patient's plasma as the blank and as the matrix for the construction of a patient-specific formate calibration curve. The blank contains all components of the assay system except the formate dehydrogenase. Formate concentration is determined from the calibration curve, constructed by adding known quantities of sodium formate to the plasma base, and plotting the fluorescence intensity against formate concentration. The assay which is sensitive to a formate level of 7 mg/L should find application in cases where formate is a metabolite.

Incidence of variants of human red cell NADH diaphorase in Japanese newborns and coordinate developmental changes of its activity with fetal hemoglobin and F cells.

615 human newborns were examined for detection of polymorphism of red cell NADH diaphorase by isoelectric focusing on polyacrylamide gels. Although the incidence of variant forms among Japanese is about 0.46%, no variants were encountered in newborns. Enzyme activity of red cell NADH diaphorase from newborns was less than half of that from adults. There were no differences in enzyme activity between premature and full-term newborns. Increase of enzyme activity during development after birth was admitted in parallel with decrease of fetal hemoglobin (Hb F) content and also with reduction of F cells (which produce only Hb F) number.

Glutathione reductase deficiency and platelet dysfunction induced by 1,3-bis(2-chloroethyl)-1-nitrosourea.

Human platelets exposed in vitro to increasing amounts of BCNU rapidly develop a progressive, relatively selective, and almost complete deficiency of GSSG-R activity. Several other enzymes are not inhibited when intact platelets are exposed to the nitrosourea; lipoamide dehydrogenase was investigated because of the remarkable similarity of the structure of its active site with that of GSSG-R. BCNU inhibits lipoamide dehydrogenase and GSSG-R only when they are in the reduced state; in the intact platelet, lipoamide dehydrogenase (unlike GSSG-R) is oxidized and is therefore unaffected. This is the first documentation of lipoamide dehydrogenase activity in platelets. After BCNU exposure, there is a reduced release of 14C-serotonin in response to collagen; the cells become incapable of aggregating in response to even large doses of epinephrine, ADP, collagen, or arachidonic acid, with loss of both primary and secondary waves of aggregation. At higher doses of BCNU, there is also a diminished PF-3 activity of intact platelets; sonication of drug-treated platelets normalizes coagulant activity. The drug-induced functional abnormalities occur despite preservation of the number of platelets, their electron microscopic appearance, and their capacity to take up 14C-serotonin. BCNU induced GSSG-R deficiency precedes the development of the earliest evidence of platelet dysfunction, and almost all of the enzyme's activity must be abolished before any functional abnormality becomes detectable. A small fraction of GSSG-R activity is essential for platelet function, and BCNU provides a powerful new tool to investigate the role of the enzymatic reduction of glutathione in platelet physiology and pathology.