Mitochondrial fixation for the detection of cytochrome oxidase activity using microwave irradiation

We examined cell fixation with microwave irradiation (MWI) used in cytochemistry. MWI was applied to blocks of about 1 mm3 of mouse parotid glands at 500 W for about 5 sec in a fixative at 37 degrees C. The activities of endogenous peroxidase and mitochondrial cytochrome oxidase were demonstrated by using the DAB method with 3,3'-diaminobenzidine (DAB) and 0.01% H2O2. Under electron microscopy, peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum and secretory granules. However, mitochondria cytochrome oxidase activity seemed to be rather weak against the MWI at 37 degrees C. Moreover, suspension of isolated hamster liver mitochondria was fixed by MWI and also demonstrated cytochrome oxidase activity by using the cytochemical methods with DAB, cytochrome c, catalase and sucrose. Such mitochondrial fractions were subjected to 6-second MWI given 10 or 18 times with an interval of 10 seconds with and without a chilled water bath. The final temperature of each fixative was kept at about 10 degrees C or rose to about 37 and 55 degrees C. When we took care to keep the temperature below 10 degrees C, the DAB reaction products accumulated in the mitochondrial intermembrane-intracristal space. No mitochondrial deposits were observed when the temperatures of the fixatives rose to 37 and 55 degrees C. These results indicated that peroxidase was very resistant to the heat with MWI fixation. Cytochrome oxidase is sensitive to the heat with MWI, so, a chilled water bath had to be used.     

A protein-specific monoclonal antibody to rat liver beta 1-->4 galactosyltransferase and its application to immunohistochemistry

BACKGROUND: The co-occurrence of panic disorder and major depression in the same individual is common. A question to be answered is whether the comorbid disorder is a distinct one or may resemble one or other disorder. In this paper we examine whether the comorbid disorder is a distinct condition. METHOD: We examined the symptom profiles and rates of comorbidity of panic attacks and DIS/DSM-III major depressive disorder in a population-based sample from four sites of the National Institute of Mental Health (NIMH) Epidemiologic Catchment Area Program (n = 12,668). RESULTS: The co-occurrence of panic attacks and major depression over the lifetime was 11 times higher than expected by chance (OR = 11.4, 95% CI 9.5 to 13.6). Subjects with both panic and depression had worse symptoms than those who had only one disorder. However, the pattern of symptoms was remarkably similar, after overall severity was taken into account. Depressive symptoms associated with more severe forms of depression (e.g. guilt, suicidal thoughts or attempts, and motor disturbance) were more frequent in the comorbid group. CONCLUSIONS: These findings may indicate a worse severity when the two disorders occur in the same individual.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase

Mutation to the conserved Glu399 or Lys192 caused the rate-limiting step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurley, T. D., and Weiner, H. (1997) J. Biol. Chem. 272, 18817-18822). Here we further investigated the role of these two NAD+-ribose-binding residues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase activity when compared with the native enzyme. No pre-steady state burst of NADH formation was found with the E399Q/K and K192Q enzymes when propionaldehyde was used as the substrate; furthermore, each mutant oxidized chloroacetaldehyde slower than propionaldehyde, and a primary isotope effect was observed for each mutant when [2H]acetaldehyde was used as a substrate. However, no isotope effect was observed for each mutant when alpha-[2H]benzaldehyde was the substrate. A pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants. The rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates. The K192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.     

Effect of glutathione depletion on the conversion of xanthine dehydrogenase to oxidase in rat liver

The ability of endogenous glutathione (GSH) to modify the activity of the enzyme xanthine oxidase (XO) in rat liver was investigated. The effect of hepatic GSH depletion on the conversion of xanthine dehydrogenase (XDH) (EC 1.1.1.204) to XO (EC 1.1.3.22) was determined 10 min after i.p. administration of different amounts of diethylmaleate to fasted rats. After administration of 400 mg/kg, total hepatic non-protein GSH (reduced + oxidized GSH) decreased significantly to 14% of controls. In this condition the level of oxidized GSH was unchanged and no lipid peroxidation was observed, while a significant increase of reversible XO and a minor increase of the irreversible form of the enzyme was detected.     

The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.     

Modification of the effects of microwave irradiation on biochemical processes by using foreign protein

The biochemical data on the influence of electromagnetic fields in microwave range on the hormonal-mediator regulation systems are presented. The possibility of biological effects modification under combined action of microwave radiation and foreign protein is discussed.     

微波烧结粗晶低钴YG硬质合金存在的脱碳问题及其改进

采用常规微波烧结法制备WC-Co硬质合金时,表层区域出现严重的脱碳现象,导致表层和中心区域的组织显著不同,即产生核壳结构,对合金的力学性能造成不利影响。本文作者以WC粉和Co粉为原料粉末,采用微波烧结法制备88%WC-12%Co(YG12)和94%WC-6%Co(YG6)硬质合金,在混料时添加炭黑,避免合金中脱碳相的生成。检验表明:当炭黑添加量(质量分数)接近0.2%时,YG12和YG6的抗弯强度(TRS)分别达到3 109和2 642 MPa;硬度(HRA)分别为88.7和89.8。此时,合金表面和中心区域具有一致的显微组织结构,没有发现脱碳相η(W3Co3C)。但当炭黑添加量超过0.2%时,大量析出的石墨相对合金的力学性能,尤其对硬度产生不利影响,当炭黑添加量为0.4%时,YG12和YG6的抗弯强度分别只有2 465 MPa和2 213 MPa。     

The application of rigid internal fixation to the treatment of non-union and delayed union using the AO technique

This paper describes our experience over a period of 10 years (1966-76) with fractures showing non-union and delayed union. All were treated by the AO technique. The source of our patients, the management of their fractures and the results are described. From this experience we have drawn conclusions on which future policy will be based.     

A method for measurement of angiotensin II in tissues and its application to rat kidney

Two distinct satellite DNAs, amounting to 25% of the total DNA, were isolated from the nuclei of the red-necked wallaby, Macropus rufogriseus. The physical properties of native, single-stranded and reassociated molecules were studied in buoyant-density gradient centrifugation. The homogeneity of each satellite fraction was examined using melting characteristics of native and reassociated DNA, and renaturation kinetics. These data suggest that sequence heterogeneity exists in both fractions. Each satellite fraction was found by in situ hybridization to be localized in heterochromatin of interphase nuclei and in the centromeric regions of metaphase chromosomes. The chromosomal distributions of the two satellite DNAs differentiate the sex chromosomes, which have sequences of only one satellite, from the autosomes which have sequences of both satellites in the centromeric heterochromatin. Giemsa C-banding techniques also showed a differentiation of the centromeric regions of sex chromosomes from those of the autosomes.     

High-performance liquid chromatographic method for the determination of norfloxacin glutamate and glucuronate in solid and liquid dosage forms and its application to stability testing

A simple, precise, stability-indicating reversed-phase high-performance liquid chromatographic method for norfloxacin glutamate and norfloxacin glucuronate in liquid and solid dosage forms is described. Chloronitrodiazepine was used as the internal standard. The eluent used with a C18 bonded phase column was methanol-water-diethylamine (50:50:0.4, v/v/v) (pH* 5.5). The effects of the eluent pH*, the ratio of methanol to water, and the quantity of diethylamine on the retention times of the sample and internal standard were investigated. The method showed good linearity in the range 1-45 micrograms ml-1 for norfloxacin. Solid samples were ground, dissolved in the eluent, filtered, and then determined by this method. Liquid samples were dissolved in the same solvent. The average recoveries of norfloxacin glutamate and norfloxacin glucuronate in their simulated preparations were 99.5% for solid products and 99.8% for liquid products. The method was applied to the study of the thermal stability of the drugs by following the degradation of norfloxacin glutamate and glucuronate in the four products in accelerated tests at 37-80 degrees C for up to 3 months. Shelf-lives at 25 degrees C of the four products were predicted from the results assuming zero- and first-order kinetics of decomposition, and were at least 1.5 years for liquid products and 2 years for solid products.     

In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.     

Bioactivation of estrone and its catechol metabolites to quinoid-glutathione conjugates in rat liver microsomes

Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the present study, we explored factors which influence the rate of P450-catalyzed formation of the o-quinones (3,5-cyclohexadiene-1,2-diones) from 2-hydroxyestrone (2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver microsomes. The initially formed o-quinones were trapped as their GSH conjugates which were separated and characterized by HPLC with electrospray-MS detection. Two mono-GSH conjugates were observed from the 2-OHE-o-quinone as well as a conjugate where GSH had added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-quinone gave only one mono-GSH adduct as well as a di-GSH adduct. Both 2-OHE and 4-OHE were excellent substrates for P450, generating o-quinone GSH adducts at 94 and 40 times, respectively, the rate of estrone. 2-OHE but not 4-OHE saturated P450 at unusually low concentrations (0.2 nmol of P450/mL) perhaps due to differences in the stability of the o-quinones formed in the active site of the enzyme. Preliminary data suggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to quinone methides (4-alkyl-2,5-cyclohexadien-1-ones, QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determined. The o-quinone from 2-OHE has a half-life of 42 +/- 3 s at 37 degrees C (pH 7.4), and the o-quinone from 4-OHE has a half-life of 12.2 +/- 0.4 min under identical conditions. The o-quinones of the AB ring analogs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene) isomerize to QMs, suggesting that a similar reaction pathway could occur with the o-quinones from catechol estrogens. In support of this, oxidation of 4-OHE and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestrone (3-hydroxy-1,3,5-(10),9(11)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM--glutathione conjugate, both of which could eliminate to give the conjugated alkene of 4-OHE. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.     

Identification of a peptide of the guanosine triphosphate binding site within brain glutamate dehydrogenase isoproteins using 8-azidoguanosine triphosphate

Photoaffinity labeling with [gamma-32P]8N3GTP (8-azidoguanosine triphosphate) was used to identify the guanine binding peptides of the GTT binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N3GTP, without photolysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities. Saturation of photoinsertion of GDH isoproteins revealed an apparent Kd of 8 microM (GDH I) and 24 microM (GDH II) for [gamma-32P]8N3GTP. Ion exchange and reversed-phase high-performance liquid chromatography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine binding domain within the GTP binding site is the region containing the sequence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M E-R (GDH I) and I-S-G-A-S-E-X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as a photolabeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site. Photolabeling of these peptides was prevented by the presence of GTP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptide identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.     

Calibration and standardization of microwave ovens for fixation of brain and peripheral nerve tissue

BACKGROUND: The role of the inhibitory neurotransmitter gamma aminobutyric acid (GABA) in schizophrenia has previously been investigated using postmortem material. Recently, using single photon emission tomography (SPET) with the selective benzodiazepine antagonist 123I-Iomazenil as the radioligand, we have demonstrated an in vivo relationship between reduced GABAA/benzodiazepine receptor binding and the severity of positive symptomatology in schizophrenia. The present study aimed to build on this using the same in vivo scanning techniques, and relating findings to cognitive functioning. METHODS: Ten nonpsychiatric control subjects and 15 schizophrenic patients, matched for age and handedness, were scanned. A battery of neuropsychologic tests was also administered. RESULTS: Correlational analysis revealed a pattern of increased correlations between GABAA/benzodiazepine receptor binding and task performance, in the schizophrenic group compared to the control group. CONCLUSIONS: Findings are preliminary but suggest a relationship between reduced GABAA/benzodiazepine receptor binding and poorer cognitive functioning, involving memory and visual attention processes, in the schizophrenic group but not in the control group. A role for GABA in the pathophysiology of schizophrenia is suggested. Limitations of the present study and suggestions for future research are discussed.     

Detection of benzoquinone adducts to rat liver protein sulfhydryl groups using specific antibodies

Benzoquinone is an electrophilic metabolite of bromobenzene and other simple aromatic compounds of toxicological interest including benzene, phenol, hydroquinone, and acetaminophen. In reacting with proteins benzoquinone shows great selectivity for Michael addition to sulfhydryl groups and formation of S-(2,5-dihydroxyphenyl) protein adducts. To facilitate the specific detection and eventual isolation and identification of such adducted proteins, we prepared an antiserum capable of recognizing hydroquinone moieties by immunizing rabbits with keyhole limpet hemocyanin modified with 3-[2,5-dihydroxyphenyl)thio]propanoyl groups as haptens. The antiserum had a high titer and showed high specificity for hapten in competitive ELISA with hapten analogues. In Western blot experiments the antiserum detected not only synthetically haptenized control proteins but also several proteins from rat liver microsomes that had been incubated in vitro with [14C]bromobenzene. This binding was completely blocked by free hapten, showing that it was hapten-specific. Each of the microsomal protein bands detected in the Western blots also contained radioactivity, but not all radioactive protein bands reacted with antibody. This antiserum should prove useful in exploring the role of protein arylation by benzoquinone in cytotoxic responses to its metabolic precursors.     

Assessment of the longevity of the liver using a rat transplant model

BACKGROUND: Our goal was to study the long-term follow-up of patients having aortic valve replacement and to focus particularly on the patients receiving small prostheses. METHODS: Four hundred twenty-eight Medtronic-Hall valves were implanted (156 size 20 or 21 mm, 149 size 22 or 23 mm, and 123 size 25 or 27 mm). Group 20-21 had a higher number of female patients, more associated coronary lesions, and more patients with aortic stenosis. RESULTS: The actuarial survival rate at 8 years was 80% for group 20-21, 80% for group 22-23, and 76% for group 25-27 (p = not significant). In group 20-21, the actuarial event-free rates at 8 years were as follows: thromboembolic complications, 94%; prosthetic valve endocarditis, 99%; reoperation, 98%; and hemorrhagic complications, 78%. The only factors of prognostic value in this group were age and associated coronary lesions. CONCLUSIONS: The durable nature of the results obtained with the Medtronic-Hall 20- and 21-mm prostheses compared with large-diameter prostheses allows the use of a simple and reliable surgical technique and should mean that indications for ring enlargement become rare.     

Responsiveness of rat nonparenchymal liver cells and spleen cells to the decomposition of erythrocytes exposed to di-n-butyl phthalate and its metabolite

We have reported that di-n-butyl phthalate (DBP) caused the depletion of circulating iron, characterized by the release of iron from both haemoglobin (Hb) and transferrin (Tf). The present study investigated whether the erythrocytes from DBP-treated rats were destroyed by nonparenchymal liver cells (NPC, including Kupffer cells) or spleen cells (SC). In the in vivo study, there were observed depletions of Hb in the blood and of iron in the hepatic Tf fraction, as well as an accumulation of iron in the hepatic hemosiderin (Hs) and splenic Tf fractions. In the in vitro study, mono-butyl phthalate (MBP), a metabolite of DBP, caused a depletion of iron in the plasma Tf, although a direct release of iron from Tf was not detectable. When erythrocytes from DBP-treated rats and erythrocytes preincubated with MBP both were incubated with NPC, respectively, the Hb was decomposed and the iron also accumulated in the cell debris. However, when the two kinds of erythrocytes were incubated with SC, respectively, no decomposition of Hb was observed at low and medium doses, but the highest dose induced an accumulation of iron to Tf. Therefore, the NPC may contribute in part to the decomposition of DBP- or MBP-affected erythrocytes.