Fabrication of a liquid-gated enzyme field effect device for sensitive glucose detection.

This study presents fabrication of a liquid-gated enzyme field effect device and its implementation as a glucose biosensor. The device consisted of four electrodes on a glass substrate with a channel functionalized by carboxylated multi-walled carbon nanotubes-polyaniline nanocomposite (MWCNTCOOH/PAn) and glucose oxidase. The resistance of functionalized channel increased with increasing the concentration of glucose when an electric field was applied to the liquid gate. The most effective and stable performance was obtained at the applied electric field of 100 mV. The device resistance, R, exhibited a linear relationship with the logarithm of glucose concentration in the range between 0.005 and 500 mM glucose. The detection limit (S/N = 3) for glucose was about 0.5 µM. Large effective area and good conductivity properties of MWCNTCOOH/PAn nanocomposite were the key features of the fabricated sensitive and stable glucose biosensor.

Nano-sized manganese-calcium cluster in photosystem II.

Cyanobacteria, algae, and plants are the manufacturers that release O2 via water oxidation during photosynthesis. Since fossil resources are running out, researchers are now actively trying to use the natural catalytic center of water oxidation found in the photosystem II (PS II) reaction center of oxygenic photosynthetic organisms to synthesize a biomimetic supercatalyst for water oxidation. Success in this area of research will transcend the current bottleneck for the development of energy-conversion schemes based on sunlight. In this review, we go over the structure and function of the water-oxidizing complex (WOC) found in Nature by focusing on the recent advances made by the international research community dedicated to achieve the goal of artificial water splitting based on the WOC of PS II.

Prussian blue modified glassy carbon electrodes-study on operational stability and its application as a sucrose biosensor.

Stabilisation of electrochemically deposited Prussian blue (PB) films on glassy carbon (GC) electrodes has been investigated and an enhancement in the stability of the PB films is reported if the electrodes are treated with tetrabutylammonium toluene-4-sulfonate (TTS) in the electrochemical activation step following the electrodeposition. A multi-enzyme PB based biosensor for sucrose detection was made in order to demonstrate that PB films can be coupled with an oxidase system. A tri-enzyme system, comprising glucose oxidase, mutarotase and invertase, was crosslinked with glutaraldehyde and bovine albumin serum on the PB modified glassy carbon electrode. The deposited PB operated as an electrocatalyst for electrochemical reduction of hydrogen peroxide, the final product of the enzyme reaction sequence. The electrochemical response was studied using flow injection analysis for the determination of sucrose, glucose and H(2)O(2). The optimal concentrations of the immobilisation mixture was standardised as 8U of glucose oxidase, 8U of mutarotase, 16U of invertase, 0.5% glutaraldehyde (0.025mul) and 0.5% BSA (0.025mg) in a final volume of 5mul applied at the electrode surface (0.066cm(2)). The biosensor exhibited a linear response for sucrose (4-800muM), glucose (2-800muM) and H(2)O(2) (1-800muM) and the detection limit was 4.5, 1.5 and 0.5muM for sucrose, glucose and H(2)O(2), respectively. The sample throughput was ca. 60 samples h(-1). An increase in the operational and storage stability of the sucrose biosensor was also noted when the PB modified electrodes were conditioned in phosphate buffer containing 0.05M TTS during the preparation of the PB films.

Sequential flow injection analysis of ammonium and nitrate using gas phase molecular absorption spectrometry.

A flow injection method on the basis of gas phase molecular absorption is described for the sequential determination of ammonium and nitrate. Two hundred microliters of sample solution is injected into the flow line. For ammonium determination, the sample zone is directed to a line in which reacts with NaOH (13M) and produces ammonia. But for nitrate determination, the sample zone is passed through the on-line copperized zinc (Zn/Cu) reduction column and produces ammonium ion and in the follows ammonia. The produced ammonia in both cases is purged into the stream of N(2) carrier gas. The gaseous phase is separated from the liquid phase using a gas-liquid separator and then is swept into a flow through cell, which has been positioned in the cell compartment of an UV-Vis spectrophotometer. The absorbance of the gaseous phase is measured at 194nm. Under selected conditions for sequential analysis of ammonium and nitrate, linear relations were found between the peak heights of absorption signals and concentrations of ammonium (10-650mugml(-1)) and nitrate (20-800mugml(-1)). The limit of detections for ammonium and nitrate analysis were 8 and 10mugml(-1), respectively. The relative standard deviations of repeated measurements of 50mugml(-1) of ammonium and nitrate were 2.0, 2.9%, respectively. Maximum sampling rate was about 40 samples/h. The method was applied to the determination of ammonium in pharmaceutical products and the sequential determination of ammonium and nitrate in spiked water samples.

Flow injection analysis of nitrite by gas phase molecular absorption UV spectrophotometry.

A flow injection method on the basis of gas phase molecular absorption is described for the determination of nitrite in the aqueous solution. 200 mul of nitrite solution is introduced into a carrier stream of distilled water. The carrier stream containing nitrite zone is reacted with a stream of hydrochloric acid (2 M). The stream is then segmented by O(2) gas. The produced gaseous products are purged into the O(2) segments, react with O(2) and are carried toward the gas-liquid separator. The gaseous phase is separated from the liquid stream by the use of home-made gas-liquid separator and then is swept into a home-made flow cell. The absorbance of gaseous phase is measured at 205 nm using a UV/VIS spectrophotometer. Under selected conditions, two linear ranges, up to 1000 mug ml(-1) and 1000-2000 mug ml(-1) of nitrite were obtained. The limit of detection was 7.5 mug ml(-1) NO(2)(-). The relative standard deviations of repeated measurements of 100 and 500 mug ml(-1) NO(2)(-) were 3.7 and 1.0%, respectively. Up to 30 samples h(-1) can be analyzed. Interferences in the proposed method were few and were readily overcome. The proposed method was successfully applied to the determination of nitrite in the spiked water samples, a number of meat products and urine.

Flow-injection analysis of nitrate by reduction to nitrite and gas-phase molecular absorption spectrometry.

Two flow-injection manifolds have been investigated for the determination of nitrate. These manifolds are based on the reduction of nitrate to nitrite and determination of nitrite by gas-phase molecular absorption spectrophotometry. Nitrate sample solution (300 microL) which is injected to the flow line, is reduced to nitrite by reaction with hydrazine or passage through the on-line copperized cadmium (Cd-Cu) reduction column. The nitrite produced reacts with a stream of hydrochloric acid and the evolved gases are purged into the stream of O2 carrier gas. The gaseous phase is separated from the liquid phase using a gas-liquid separator and then swept into a flow-through cell which has been positioned in the cell compartment of an UV-visible spectrophotometer. The absorbance of the gaseous phase is measured at 204.7 nm. A linear relationship was obtained between the intensity of absorption signals and concentration of nitrate when Cd-Cu reduction method was used, but a logarithmic relationship was obtained when the hydrazine reduction method was used. By use of the Cd-Cu reduction method, up to 330 microg of nitrate was determined. The limit of detection was 2.97 microg nitrate and the relative standard deviations for the determination of 12.0, 30.0 and 150 microg nitrate were 3.32, 3.87 and 3.6%, respectively. Maximum sampling rate was approximately 30 samples per hour. The Cd-Cu reduction method was applied to the determination of nitrate and the simultaneous determination of nitrate and nitrite in meat products, vegetables, urine, and a water sample.

Co-expression of CD56 and CD30 in lymphomas with primary presentation in the skin: clinicopathologic, immunohistochemical and molecular analyses of seven cases.

BACKGROUND: Natural killer and natural killer-like T-cell lymphomas presenting in the skin usually demonstrate aggressive behavior, an angiocentric distribution and a characteristic immunophenotype. In contrast, primary cutaneous CD30+ lymphoproliferative disorders form a heterogeneous spectrum including anaplastic large cell lymphomas, the majority of which display a good prognosis. Lymphomas with co-expression of CD56 and CD30 are extremely rare and the significance of this co-expression is unknown. METHODS: Seven retrospectively identified cases of lymphomas with co-expression of CD56 and CD30 presenting in the skin comprise this study. Immunohistochemistry, in situ hybridization for Epstein-Barr virus and T-cell receptor gene rearrangement studies were performed on paraffin sections. RESULTS: This subset of cutaneous lymphomas showed a variable clinical course that ranged from resolution without treatment, treatment-failure and recurrence, to death from disease. Histologic, immunophenotypic and molecular studies were of limited utility in predicting prognosis. CONCLUSIONS: Cutaneous lymphomas co-expressing CD56 and CD30 share many clinicopathologic features with natural killer and natural killer-like T-cell lymphomas or anaplastic large cell lymphomas, two entities with widely disparate clinical behavior. It is important to recognize that these lymphomas may behave more aggressively than primary cutaneous anaplastic large cell lymphomas do. Longer follow-up and further investigations on larger numbers of cases are necessary to fully characterize this rare subset of cutaneous lymphomas.

Pagetoid reticulosis (Woringer-Kolopp disease): an immunophenotypic, molecular, and clinicopathologic study.

Pagetoid reticulosis (PR), also known as Woringer-Kolopp disease, is a form of cutaneous T-cell lymphoma that demonstrates striking epidermotropism on histologic examination. We present the histologic, immunologic, and molecular findings for seven patients who had PR. The patients ranged in age from 33 to 67 years. All patients presented with one or several thick plaques involving the distal extremities except for one patient, who presented with a tongue lesion. Immunohistochemical staining of the atypical lymphoid cells demonstrated a T-cell phenotype in all cases. In one of four frozen cases, the neoplastic cells were of T-helper cell phenotype (CD4 positive). Four of seven cases demonstrated a T-cytotoxic/suppressor cell phenotype (CD8 positive). The T-cell subset for the remaining two cases could not be determined. CD30 positivity and a high growth fraction as indicated by staining with Ki-67 were seen in three of seven and three of four cases, respectively. Genotypic analysis performed on three of our cases revealed T-cell receptor (gamma and/or beta) rearrangement, indicating a clonal proliferation. The clinical follow-up ranged from 15 months to 13 years. Four of seven patients are alive and free of disease after treatment with excision or local irradiation. One patient relapsed twice after treatment with radiation and photochemotherapy with 8-methoxypsoralen and UVA and was then lost to follow-up. The lesions of another patient resolved spontaneously but recurred at the same and in an additional site 5 years later. One patient recurred after electron beam therapy. The recurrent lesion improved with radiation therapy and local wound care but never resolved completely. The patient died of unrelated causes. Our findings suggest that PR is a distinct clinicopathologic entity, separate from unilesional mycosis fungoides, demonstrating a slow disease course. The disease is a clonal cutaneous T-cell lymphoma with relatively consistent clinical and histopathologic findings but a heterogeneous immunophenotypic profile.

IgM myeloma: case report with immunophenotypic profile.

Immunoglobulin (Ig)M myeloma is a distinct subtype of multiple myeloma (MM) displaying clinical and pathologic features of both MM and Waldenström's macroglobulinemia (WM). Although the immunophenotypic characteristics of classic MM and WM have been reported, the surface antigen expression of IgM myeloma has not been reported. We report a case of IgM myeloma and describe its immunophenotypic profile using flow cytometry. The cells showed a hybrid MM-WM phenotype, strongly expressing CD38 but lacking CD45 and DR, typical for plasma cells; however, pan-B cell antigens CD20 and FMC7 as well as weak monoclonal surface Ig also were positive, resembling B-cell lymphoproliferative malignancies. Discordant B-cell antigen expression was present, in that pan-B antigens CD19 and CD22 were absent. In addition, B-cell activation antigen CD23, early B-precursor antigen CD10, and pan-T antigen CD5 were not expressed. Although CD20 and weak surface Ig expression have been reported in MM, FMC7 positivity has not been seen. The data therefore suggest that IgM myeloma may have a unique phenotype with characteristics of both MM and WM.

Flow injection analysis of sulphite by gas-phase molecular absorption UV/VIS spectrophotometry.

A flow injection gas-phase molecular absorption spectrophotometric method is described for the determination of sulphite in aqueous solution. The sulphite solution, 200 microl, is introduced into a stream of distilled water. The carrier stream containing a sulphite zone is reacted, in the first mixing coil, with a stream of sulphuric acid (1 M). The evolved sulphur dioxide is purged to the segments of nitrogen flow through the second mixing coil. The gaseous phase is separated from the liquid stream by the use of a purpose built gas-liquid separator and then is swept into a purpose built flow-through cell. The absorbance of the gaseous phase is measured at 200 nm using a UV/VIS spectrophotometer. Up to 440 microg of sulphite is determined. The limit of detection is 0.8 microg and the R.D.S. for the determination of 70 and 220 microg of sulphite are 1.02 and 0.76%, respectively. Up to 40 samples h(-1) can be analyzed. The effect of several anions and cations on the determination of sulphite was studied and the results showed that the method is relatively free from interferences. The proposed method was applied to the determination of sulphite in a synthetic sample, water sample and lemon juice.

Immunohistochemical detection of aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and nontumorous liver tissue.

Monoclonal antibodies recognizing the stable imidazole ring-opened form of the major N7-guanine aflatoxin B1-DNA adduct have been used in competitive enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays to quantitate adduct levels in liver tissue. Methods were developed in AFB1-treated animals, then applied to paired tumor and nontumor liver tissues of hepatocellular carcinoma patients from Taiwan. An avidin-biotin complex staining method was also used for of the detection of hepatitis B surface (HBsAg) and X (HBxAg) antigens in liver sections. A total of 8 (30%) hepatocellular carcinoma (HCC) samples and 7 (26%) adjacent nontumor liver tissue samples from Taiwan were positive for AFB1-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22 (81%) adjacent nontumorous liver samples were positive, and 9 (33%) HCC samples and 11 (41%) adjacent nontumor liver samples were HBxAg positive. No association with AFB1-DNA adducts was observed for HBsAg and HBxAg. These methods should be useful in determining the role of exposure in the induction of HCC in Taiwan.

Aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and non-tumorous liver tissue.

Studies were carried out to test the hypothesis that exposure to aflatoxin B1 (AFB1) is common among individuals with hepatocellular carcinoma (HCC) who are also chronically infected with hepatitis B virus (HBV). Experiments were also carried out to determine whether there is a close association between the presence of AFB1-DNA adducts and the expression of one or more HBV antigens in the tumor or non-tumor regions of the liver. Twenty-seven paired tumor and non-tumor liver tissues of HCC patients from Taiwan were analyzed. Monoclonal antibody 6A10, generated against the imidazole ring-opened persistent form of the major N-7 guanine adduct of AFB1, was used for adduct detection by both indirect immunofluorescence and competitive enzyme-linked immunosorbent assay. An avidin-biotin complex staining method was used for the detection of HBsAg and HBxAg in liver sections. A total of 8 (30%) HCC samples and 7 (26%) adjacent non-tumor liver tissue samples from Taiwan were positive for AFB1-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22 (81%) adjacent non-tumorous liver samples were positive while 9 (33%) HCC samples and 11 (41%) adjacent non-tumor liver samples were HBxAg-positive. No association with AFB1-DNA adducts was observed for HBsAg and HBxAg. These results suggest that both AFB1 exposure and carrier status of HBsAg/HBxAg may be involved in the induction of HCC in Taiwan.

Quantitation of aflatoxin B1-DNA adducts in woodchuck hepatocytes and rat liver tissue by indirect immunofluorescence analysis.

A quantitative indirect immunofluorescence technique was developed utilizing a monoclonal antibody (6A10) recognizing the imidazole ring-opened form of the major N-7 guanine adduct of aflatoxin B1 (AFB1). This method was used to investigate adduct formation in woodchuck hepatocytes treated in culture and in liver tissue of rats treated i.p. with AFB1. Fluorescein isothiocyanate-labeled secondary antiserum was used for adduct localization in conjunction with 4',6-diamidino-2-phenylindole dihydrochloride staining to localize nuclei. Quantitation of AFB1-DNA adducts was carried out by densitometric analysis of photographic slides. Specific nuclear staining was observed in both woodchuck hepatocytes and rat liver tissue. There was a dose-response relationship between fluorescence intensity and AFB1 dose in treated animals. Turnover of adducts could also be followed in animals over 48 h with this method. DNA was isolated from liver tissue of treated animals and adduct levels were quantitated by competitive enzyme-linked immunosorbent assay with antibody 6A10 and by fluorescence spectroscopy. There was a significant correlation of the quantitative immunofluorescence intensity with levels of AFB1 adducts detected by enzyme-linked immunosorbent assay (r = 0.61, P less than 0.05) and spectrofluorescence (r = 0.78, P less than 0.01). This immunohistochemical method should be applicable to the detection of adducts in liver tissues of humans exposed to high levels of dietary AFB1.

Inhibitory effect of epinephrine on phosphatidate activity in isolated rat hepatocytes.

The variations in phosphatidate phosphohydrolase activity were investigated in the post-mitochondrial fraction of isolated rat hepatocytes incubated for short periods with epinephrine, dibutyryl cyclic AMP or oleate. Epinephrine decreased the enzyme activity by 42% at 1 microM concentration. The inhibitory effect was abolished in the presence of a beta-adrenoceptor antagonist, propranolol, but was not affected by the alpha-adrenoceptor agonist, phenylephrine, or the agonist, phentolamine. Dibutyryl-cAMP inhibited the enzyme activity by 49%. The presence of the cyclic-AMP phosphodiesterase inhibitor, aminophyline, together with epinephrine slightly increased the enzyme inhibition. Oleate stimulated the enzyme activity (100%) and its effect was antagonized by dibutyryl-cyclic AMP (24%) and by epinephrine (15%). The results indicate that epinephrine acts on rat hepatocytes via beta-adrenoceptor activation and cAMP may be involved in the mechanism by which phosphatidate phosphohydrolase is regulated.

The effects of hydrazine on the phosphatidate phosphohydrolase activity in rat liver.

Injection of hydrazine (0.7 mmole/kg) in the male fasting rats caused an increase in phosphatidate phosphohydrolase (PAP) activity in the soluble fraction of the liver. The increased PAP activity was parallel with a rise in hepatic triacylglycerol (TG) (3.5-fold) and in the catecholamine concentration (3.4-fold) in adrenal glands. Hydrazine also increased serum glucose. The hydrazine-induced increase in PAP activity and TG accumulation was completely prevented by adrenalectomy. The data suggest that increased PAP activity is at least partly responsible for hydrazine-induced fatty liver and that adrenal hormones may take part in the mechanism by which hydrazine exerts its effects on the liver.

A genetic variant of human erythrocyte glucose 6-phosphate dehydrogenase.

Human erythrocyte G6PD activity was measured in more than 500 subjects in Isfahan, Iran, and the percent of enzyme deficiency for males and females are reported. Some properties of the abnormal enzyme is compared with its normal counterpart. Apparent Km values of glucose 6-phosphate for the variant and normal enzymes were 37 and 101 microM, respectively. The variant enzyme was less resistant to inhibition by 40 microM NADPH (72% inhibition) than the normal enzyme (48% inhibition). The mode of inhibition for both enzymes was competitive with NADP+. ATP at 1.5 mM concentration also inhibited normal and variant enzymes at 17% and 10%, respectively. The inhibition was competitive with glucose 6-phosphate. Polyacrylamide gel electrophores showed that normal enzyme has one major and another weak active bands, while the variant enzyme under identical conditions shows only one active band corresponding to the major band of the normal enzyme. Thermostability of variant G6PD was slightly lower that normal but no significant differences observed in their energy of activation. The activity pH profile of the variant enzyme was truncate.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Kinetics of reassociation and reactivation from inactive subunits.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is denatured in 8 M urea and dissociated into its two inactive subunits. Denaturation leads to an approximately 80% decrease in protein fluorescence and a 20-nm red shift in the emission maximum. Upon dilution, the urea-treated enzyme regains catalytic activity (approximately 70%). The reactivated enzyme is indistinguishable from the native enzyme based on a number of physicochemical and enzymological criteria. The kinetics of renaturation and reactivation were monitored by measuring the rates of regain of native fluorescence and appearance of activity and the accessibility of histidine residues toward diethyl pyrocarbonate modification. Regain of native fluorescence was too rapid to measure at 25 degrees C; at 5 degrees C the initial phase was also too rapid, but a slower phase was monitored and shown to obey first-order kinetics with k = (5.9 +/- 1.3) x 10(-3) s-1. Reappearance of activity was measured at several protein concentrations; reactivation followed second-order kinetics with k = (4.85 +/- 0.47) x 10(-3) M-1 min-1. Reactivation was stimulated to different degrees by either the initial or delayed addition of NAD+, NADP+, or glucose 6-phosphate. During the initial, rapid phase of renaturation, approximately 3 of the enzyme's 12 histidine residues become unreactive toward diethyl pyrocarbonate; concomitant with the subsequent reactivation, approximately 7 more histidines become inaccessible to diethyl pyrocarbonate. The data are consistent with a model for enzyme renaturation and reactivation in which the unfolded subunits rapidly refold to an inactive structure that can dimerize slowly to generate native enzyme. Specific ligands stimulate reactivation by binding to refolded subunits or incompletely folded dimers.