The mechanism of conversion of rat liver xanthine dehydrogenase from an NAD+-dependent form (type D) to an O2-dependent form (type O).

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD⁺-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O₂-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD⁺-dependent dehydrogenase molecule.     

Pulses of darkness shift the phase of a circadian rhythm in an insectivorous bat

Summary Microspectrophotometric measurements on the retina ofDeilephila elpenor were performed using fresh (cut by razor blade) as well as frozen (cryostate microtome) sections. Difference spectra from various parts of the rhabdom clearly showed that in the distal part of the rhabdom the UV pigment is predominant, while in the medial part only green pigment was found. From the difference spectra, wave-length and relative height of the absorption maxima of rhodopsins and metarhodopsins were computed and were found to be in close agreement with other results reported in the literature. These findings confirm the work of Welsch (1977) who concluded from his electron microscopic analysis that the two distal receptor cells are UV sensitive, while the six median receptor cells are green sensitive. There is also good evidence to assume that the ninth, proximal receptor is violet sensitive.We would like to thank Mrs. E. Brüning for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft SFB 114 Bionach.     

Optical Spectral Surveillance of Breast Tissue Landscapes for Detection of Residual Disease in Breast Tumor Margins

We demonstrate a strategy to “sense” the micro-morphology of a breast tumor margin over a wide field of view by creating quantitative hyperspectral maps of the tissue optical properties (absorption and scattering), where each voxel can be deconstructed to provide information on the underlying histology. Information about the underlying tissue histology is encoded in the quantitative spectral information (in the visible wavelength range), and residual carcinoma is detected as a shift in the histological landscape to one with less fat and higher glandular content. To demonstrate this strategy, fully intact, fresh lumpectomy specimens (n = 88) from 70 patients were imaged intra-operatively. The ability of spectral imaging to sense changes in histology over large imaging areas was determined using inter-patient mammographic breast density (MBD) variation in cancer-free tissues as a model system. We discovered that increased MBD was associated with higher baseline β-carotene concentrations (p = 0.066) and higher scattering coefficients (p = 0.007) as measured by spectral imaging, and a trend toward decreased adipocyte size and increased adipocyte density as measured by histological examination in BMI-matched patients. The ability of spectral imaging to detect cancer intra-operatively was demonstrated when MBD-specific breast characteristics were considered. Specifically, the ratio of β-carotene concentration to the light scattering coefficient can report on the relative amount of fat to glandular density at the tissue surface to determine positive margin status, when baseline differences in these parameters between patients with low and high MBD are taken into account by the appropriate selection of threshold values. When MBD was included as a variable a priori, the device was estimated to have a sensitivity of 74% and a specificity of 86% in detecting close or positive margins, regardless of tumor type. Superior performance was demonstrated in high MBD tissue, a population that typically has a higher percentage of involved margins.     

Selenite binding to carbon monoxide oxidase from Pseudomonas carboxydovorans. Selenium binds covalently to the protein and activates specifically the CO----methylene blue reaction

The CO----methylene blue and CO----dichlorophenol indophenol activities of carbon monoxide oxidase were specifically activated upon aerobic incubation with selenite, whereas the NADH----methylene blue activity was not altered. Fully active enzyme contained selenium, molybdenum, and flavin adenine dinucleotide in a 1:1:1 ratio. Selenium was covalently bound to the protein, probably between the sulfurs of half-cystine residues, and not a constituent of the molybdenum cofactor. The action of selenite was directed to the cytoplasmic species of carbon monoxide oxidase exclusively, whereas the CO----methylene blue activity of the membrane-bound enzyme remained unaffected.     

Changes in certain hematological and physiological variables following single gallium arsenide exposure in rats

Gallium arsenide (GaAs), a group III-VA intermetallic semiconductor, possesses superior electronic and optical properties and has a wide application in electronic industry. Exposure to GaAs in the semiconductor industries could be a possible occupational risk. The aim of the present study was to determine the dose-dependent effect of single oral exposure to GaAs (500, 1000, or 2000 mg/kg) on some biochemical variables in heme synthesis pathway and few selected physiological variables at d 1, 7, and 15 following administration. The results indicate that GaAs produced a significant effect on the activity of δ-aminolevulinic acid dehydratase (ALAD) in blood and heart (particularly at d 7) following exposure to 2000 mg/kg, whereas urinary δ-aminolevulinic acid (ALA) excretion was elevated only at d 7. No marked influence of GaAs on blood hemoglobin, zinc protoporphyrin, and packed cell volume was noticed. Blood glutathione (GSH) was significantly reduced at d 7, but remained unchanged at two other time intervals. On the other hand, heart GSH contents remained uninfluenced on GaAs exposure. Most of the physiological variables, viz. blood pressure, heart and respiration rate, and twitch response, remained unchanged, except for some minor alterations observed at d 7 and 15 following exposure to GaAs at a dose of 2000 mg/kg. Blood gallium concentration was not detectable in normal animals and rats exposed to 500 mg/kg GaAs. Blood arsenic concentration was, however, detectable even at the a lower dose level and increased in a dose-dependent manner. All these changes showed a recovery pattern at d 21, indicating that the alterations are reversible.     

Studies on SAGO wastewater treatment using anaerobic rotating biological contactor

Experimental studies were done in a laboratory scale Anaerobic Rotating Biological Contactor (RBC), for treatment of Synthetic sago wastewater. This paper describes the development and laboratory testing of an Anaerobic RBC process that couples the advantages of the fixed film horizontal flow RBC process with the high strength, starch degradation capabilities of anaerobic systems. The reactor was operated at ambient temperature and was subjected to organic and hydraulic loading rates. The reactor performance with respect to Chemical Oxygen Demand (COD) removal, alkalinity, volatile acids at each stage and biogas production were evaluated. The Anaerobic RBC reactor liquid volume is 70 litres and total disc surface area is 4.45 m². The reactor was operated with about 100% of the disc area submerged and with a rotational speed held constant at 9?rev/min. The synthetic sago wastewater was started with a COD value of 1087?mg/l at a hydraulic retention time(HRT) of 42?h and it was varied till maximum COD of 9522?mg/l. From the present study, the optimum COD load was found to be 6860?mg/l with a COD removal efficiency of 97.2%.With this optimum COD load, hydraulic loading rate(HLR) study was done at 24?h to 48?h HRT. COD removal efficiencies at hydraulic loading rates were compared with the work of Subrahmanyam &; Sastry (1988). From the present study, the proportionality coefficient was found to be 1.18 with process efficiencies at different hydraulic loading rates.     

Role of intramolecular disulfides in stability and structure of a noncovalent homodimer

The importance of intramolecular disulfides in a noncovalent dimeric protein interleukin-8 (IL-8) has been studied by replacing cysteines in each of the two disulfide pairs with alpha-aminobutyric acid (CH(2)-SH --> CH(2)-CH(3)). Both disulfide mutants are less stable and exist as molten globules in the monomeric state. Interestingly, both mutants dimerize, though with slightly lower affinities compared to the native protein. NMR studies suggest a molten globule-like structure also in the dimeric state. Structures, sequence analysis, and mutagenesis studies have shown that the conserved hydrophobic residues are packed against each other in the protein core and that H bonding and van der Waals interactions stabilize the dimer interface. Deleting either disulfide in IL-8 results in substantial loss in receptor activity, indicating that both disulfides are critical for function in the folded protein. These data together suggest that the packing interactions of the hydrophobic core determine IL-8 monomer fold, that disulfides play only a marginal role in dimer formation, and that the stability imparted by the disulfides is intimately coupled to fold and function.     

Treatment of distillery spentwash by hybrid UASB reactor

A laboratory-scale hybrid UASB reactor, which combined an UASB in the lower part and a filter in the upper part, was used for the treatment of distillery spentwash. The reactor was operated under ambient conditions for 380 days. Using anaerobically digested sewage sludge as a seed, the start-up of the reactor and the cultivation of active granular sludge was completed within three months period. Scanning electron microscopic (SEM) observation of the granules showed the presence of Mehtanonthrix-like bacteria as the dominant species. Following the start-up the organic loading rate (OLR) was increased, stepwise, to 36 kg COD/m³ · d at a constant hydraulic retention time (HRT) of 6 h. COD removal efficiency was 80% even at a high OLR of 36 kg COD/m³ · d. Biogas rich in methane content (80%), with a maximum specific biogas yield of 0.40 m³ CH₄/kg · COD was produced. Polypropylene pall rings filter medium in the upper-third of the reactor was very effective as a gas-liquid-solid (GLS) separator, and retained the biomass in addition. The study indicated that hybrid UASB is a very feasible alternative for the treatment of high-strength wastewaters like distillery spentwash.