Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

A pellet method to demonstrate nitrogen fixation by free-living rhizobia

Summary A method is described to demonstrate nitrogen fixation by free-living Rhizobium cells. After aerobic growth in a nutrient solution, the bacteria are centrifuged. Acetylene reduction by the rhizobial cells in the pellet can be measured within a few days. Hydrogen gas frequently stimulates acetylene reduction.     

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

Dihydroorotate dehydrogenase from Saccharomyces cerevisiae: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues

In all organisms the fourth catalytic step of the pyrimidine biosynthesis is driven by the flavoenzyme dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11). Cytosolic DHODH of the established model organism Saccharomyces cerevisiae catalyses the oxidation of dihydroorotate to orotate and the reduction of fumarate to succinate. Here, we investigate the structure and mechanism of DHODH from S. cerevisiae and show that the recombinant ScDHODH exists as a homodimeric enzyme in vitro. Inhibition of ScDHODH by the reaction product was observed and kinetic studies disclosed affinity for orotate (K(ic)=7.7 microM; K(ic) is the competitive inhibition constant). The binding constant for orotate was measured through comparison of UV-visible spectra of the bound and unbound recombinant enzyme. The midpoint reduction potential of DHODH-bound flavine mononucleotide determined from analysis of spectral changes was -242 mV (vs. NHE) under anaerobic conditions. A search for alternative electron acceptors revealed that homologues such as mesaconate can be used as electron acceptors.     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Comparison of Zinc Reduction with Platinum Reduction for Analysis of Deuterium‐Enriched Water Samples for the Doubly Labeled Water Technique

Objective: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium‐enriched water samples. Research Methods and Procedures: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). Results: For the zinc method, the standard deviation for multiple sets of triplicate ²H₂O sample analysis was ±4.36‰ and ±2.07‰ for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra‐assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was ?438 to 550 kcal/day. Discussion: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium‐enriched water samples. The platinum method is also less costly and less labor‐intensive than the zinc method.     

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.