Characterization of iron-sulfur clusters in rat liver submitochondrial particles by electron paramagnetic resonance spectroscopy. Alterations produced by chronic ethanol consumption

Iron-sulfur clusters present in rat liver submitochondrial particles were characterized by ESR at temperatures between 30 and 5.5 K combined with potentiometric titrations. The spectral and thermodynamic characteristics of the iron-sulfur clusters were generally similar to those previously reported for pigeon or bovine heart submitochondrial particles. Clusters N-1a, N-1b, N-2, N-3 and N-4 of NADH dehydrogenase had midpoint oxidation-reduction potentials at pH 7.5 of ?425, ?265, ?85, ?240 and ?260 mV, respectively. Clusters S-1 and S-3 of succinate dehydrogenase had midpoint potentials of 0 and +65 mV, respectively. The iron-sulfur cluster of electron-transferring flavoprotein-ubiquinone oxidoreductase exhibited the g_z signal at g = 2.08 and had a midpoint potential of +30 mV. This signal was relatively prominent in rat liver compared to pigeon or bovine heart.Submitochondrial particles from rats chronically treated with ethanol (36% of total calories, 40 days) showed decreases of 20–30% in amplitudes of signals due to clusters N-2, N-3 and N-4 compared to those from pair-fed control rats. Signals from clusters N-1b, S-1, S-3 and electron-transferring flavoprotein-ubiquinone oxidoreductase were unaffected. Microwave power-saturation behavior was similar for both submitochondrial particle preparations, suggesting that the lower signal amplitudes reflected a lower content of these particular clusters. NADH dehydrogenase activity was significantly decreased (46%), whilst succinate dehydrogenase activity was elevated (25%), following chronic ethanol consumption. The results indicate that chronic ethanol treatment leads to an alteration of the structure and function of the NADH dehydrogenase segment of the electron transfer chain. This alteration is one of the factors contributing to the lower respiration rates observed following chronic ethanol administration.     

WOMEN IN NINETEENTH CENTURY AMERICAN BOTANY; A GENERALLY UNRECOGNIZED CONSTITUENCY

Botany was thought to be a suitable study for young women in schools and an amateur avocation in the Nineteenth Century. A surprisingly large number of American women identified themselves as being seriously interested in botany. For example, in the first published directory of American botanists in 1873, 13 percent of the 599 names are women's and that increased to 16 percent of the 982 names in 1878. In this paper, 1,185 women have been identified as a sample of those actively interested in botany during the century. Less than 2% of them were active before 1870, and most of them, 67%, resided in New England and the Middle Atlantic States. Almost three quarters of them were unmarried, and only 10% had higher educational degrees although 15% had some identifiable profession. Some particular individuals are noted for their contributions to science, their activity as plant collectors, and their support of botanical societies. Though few American women became professional botanists in the Nineteenth Century, they constitute an important overlooked constituency for the developing profession of botany.     

Protein modules

As the database of protein sequences grows it is becoming apparent that many proteins are constructed from relatively few modular units that appear many times. Determination of the three-dimensional structure of such modules by NMR has been possible due to their production in relatively large quantities by recombinant DNA techniques. The knowledge gained about the structure of individual modules can then be used to predict their properties in a variety of intact proteins.     

Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.     

Mannosidase II and the 135-kDa Golgi-specific antigen recognized monoclonal antibody 53FC3 are the same dimeric protein

Monoclonal antibodies are frequently used as organelle-specific markers without identifying the specific antigen recognized. We have purified the protein recognized by a Golgi-specific monoclonal antibody 53FC3 (Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1, 1621-1628). Peptide microsequencing suggested that this antigen is mannosidase II, which was confirmed by cross-immunoprecipitation with an anti-mannosidase II antibody and by precipitation of mannosidase activity with the monoclonal antibody. Mannosidase II was found to exist normally as a disulfide-linked dimer.     

Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA

We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.     

Antimycin inhibition as a probe of mitochondrial function in isolated rat hepatocytes Effects of chronic ethanol consumption

Previous studies have established that hepatic mitochondria and submitochondrial particles from rats, fed ethanol chronically, display diminished respiratory activities and alterations in the contents of specific electron transfer chain components. The latter include a decrease of about 50% in cytochrome b content. Titrations of respiratory activity in submitochondrial particles with antimycin, a stoichiometric inhibitor of electron flow through the cytochrome b-c₁ region of the respiratory chain, indicated a comparable decrease (35%) in the amount of antimycin required to elicit maximal inhibition (‘titer’) after chronic ethanol treatment. Measurements of antimycin binding to submitochondrial particles by fluorescence quenching demonstrated a similar diminution in the number of tight binding sites per mg protein. By contrast, hepatocytes isolated from control and ethanol-fed rats exhibited nearly identical rates of oxygen utilization under a variety of conditions. However, antimycin titrations of respiratory activity in isolated hepatocytes revealed a 60% decrease in the antimycin titer, but no change in the maximal extent of inhibition after chronic ethanol treatment. Direct measurements of cytochrome b which could be reduced in the presence of antimycin in hepatocytes confirmed a comparable decrease (42%) after chronic ethanol treatment. The results demonstrate that molecular alterations in the cytochrome b region of the respiratory chain caused by ethanol feeding are present in intact liver cells, but suggest that substrate accessibility, rather than the respiratory chain, limits the rate of oxygen utilization in isolated hepatocytes. The data also suggest that mitochondria account for at least 80% of total oxygen utilization by liver cells from both control and ethanol-fed rats.