Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

Maltose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway

The hyperthermophilic anaerobe Pyrococcus furiosus was grown on maltose as energy and carbon source. During growth 1 mol maltose was fermented to 3–4 mol acetate, 6–7 mol H₂ and 3–4 mol CO₂. The presence of the following enzyme activities in cell extracts of maltose-grown P. furiosus indicate that the sugar is degraded to pyruvate and H₂ by a modified non-phosphorylated Entner-Doudoroff-pathway (the values given in brackets are specific enzyme activities at 100 °C): Glucose: methyl viologen oxidoreductase (0.03 U/mg); 2-keto-3-deoxy-gluconate aldolase (0.03 U/mg); glyceraldehyde: benzyl viologen oxidoreductase (2.6 U/mg), glycerate kinase (2-phosphoglycerate forming) (0.48 U/mg), enolase (10.4 U/mg), pyruvate kinase (1.4 U/mg). Hexokinase, glucose-6-phosphate dehydrogenase, 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphofructokinase could not be detected. Further conversion of pyruvate to acetate, CO₂ and H₂ involves pyruvate: ferredoxin oxidoreductase (0.4 U/mg; T=60°C with Clostridium pasteurianum ferredoxin as electron acceptor), hydrogen: methyl viologen ixodoreductase (3.4 U/mg) and ADP-dependent acetyl-CoA synthetase (1.9 U/mg). Phosphate acetyl transferase and acetate kinase could not be detected. The ADP-dependent acetyl-CoA synthetase catalyzes ATP synthesis via the mechanism of substrate level phosphorylation and apparently constitutes the only ATP conserving site during maltose catabolism in P. furiosus.This novel pathway of maltose fermentation to acetate, CO₂ and H₂ in the anaerobic archaeon P. furiosus may represent a phylogenetically ancient pathway of sugar fermentation.Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - BV benzyl viologen - CHES cyclohexylamino-ethane sulfonic acid - ABTS 2,2-Azino-di-(3-ethylbenzthiazoliumsulfonate)     

Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus

The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×10⁸ cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO₂ and 0.8 mol H₂ were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO₂, 1.2 mol H₂ and 0.03 mol acetoin. After fermentation of [3-¹⁴C]pyruvate the specific radioactivities of pyruvate, CO₂ and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg^-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg^-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg^-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg^-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO₂ and H₂ involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - MOPS morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany     

Chromosome localization and gene synteny of the major histocompatibility complex in the owl monkey,Aotus

Rodent cells were hybridized with owl monkey (Aotus) cells of karyotypes II, III, V, and VI. Aotus-rodent somatic hybrid lines preferentially segregating Aotus chromosomes were selected to determine the chromosomal location of the major histocompatibility complex and other genes with which it is syntenic in man. Based on correlation between concordant segregation of the chromosome as visualized by G-banding and expression of the Aotus antigens or enzymes in independent Aotus-rodent hybrid clones, we have assigned Aotus gene loci for the MHC, GLO, ME₁, SOD₂, and PGM₃ to Aotus chromosome 9 of karyotype VI (2n=49/50), chromosome 10 of karyotype V (2n=46), and chromosome 7 of karyotypes II and III (2n = 54 and 53). On the basis of banding patterns we previously postulated that these chromosomes of the different karyotypes were homologous. The gene assignments reported here provide independent evidence for that hypothesis. Aotus chromosomes 9 (K-VI), 10 (K-V), and 7 (K-II, III) are homologous to human chromosome 6 in that they all code for the MHC, GLO, ME₁, SOD₂, and PGM₃. The structural differences between these homologous chromosomes probably resulted from a pericentric inversion.Abbreviations used in this paper MHC major histocompatibility complex - HLA human lymphocyte antigen - PGM3 phosphoglucomutase-3 - ME₁ cytoplasmic malic enzyme-1 - SOD₂ superoxide dismutase-B - GLO glyoxalase 1 - OMLA owl monkey leukocyte antigens - K karyotype - ₂-M ₂-microglobulin - DMEM Dulbecco's modification of Eagle's medium - PEG polyethylene glycol - HAT hypoxanthine, aminopterin, and thymidine - KC1 potassium chloride - G-band-trypsin Giemsa band     

Factors affecting the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation

The effect of medium composition on the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation has been studied. Four types of mycelial pellets were obtained under the conditions used and may be classified as (a) frayed and loose with 0.1–0.5 mm diameter (b) compact with 0.1–0.5 mm diameter (c) loose with 0.5–2.0 mm diameter and (d) compact with 0.5–2.0 mm diameter. Their respective maximum specific rates of formation and yields of itaconic acid, based on 100 g sucrose supplied, were (a) 1.25 mol mg^–1h^–1 and 55–59 g, (b) 0.27–0.43 mol mg^–1 h^–1 and 26–38 g, (c) 0.75–0.90 mol mg^–1 h^–1 and 45–51 g and (d) 0.12 mol mg^–1 h^–1 and 10 g. The presence of Ca²⁺, Zn²⁺ and Fe²⁺ in the basal medium at concentrations of 23.3 mg/100 ml, 0.01 mg/100 ml and 0.006 mg/100 ml respectively were found to be adequate and crucial in obtaining the desired outgrowth for both high production rates and consistent yields of itaconic acid. The further addition of either commercial plaster of Paris or analytical-reagent-grade CaSO₄, especially when activated by heating to 530°C and present in excess of solubility, results in small and frayed pellets, which lead to itaconic acid yields of 55–59 g acid/100 g sugar supplied.     

Effect of exogenous plant growth regulators on in vitro seed growth,embryo development,and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.)

Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA₃ and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar Klages × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA₃ alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA₃ produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. II. Identification of allotypes Lpm-7 and Lpm-8 and genetic control of seven markers of the Lpm system

By means of alloimmunization of mink, two new antigens, Lpm-7 and Lpm-8, were detected in their sera. Lpm-7 and Lpm-8 allospecificities were referred to a very high density ₂-lipoprotein (Lpm) by the following criteria: histochemical tests, immunoelectrophoresis, preparative ultracentrifugation, and coalescence of alloprecipitates with heteroprecipitates in double diffusion tests. Genetic analysis indicated that Lpm-7 and Lpm-8, together with the earlier described Lpm-1, Lpm-2, Lpm-3, Lpm-4, and Lpm-5, share a common immunogenetic system. Polymorphism for the seven markers is conditioned by the genetic units Lpm ⁸, Lpm ⁴, Lpm ^{4, 8}, Lpm ^{4, 7}, Lpm ^{3, 4, 8}, Lpm ^{1, 8}, Lpm ^{1, 2, 7}, and Lpm ^{2, 4, 5, 7}, which behave as alleles. Of these units, the latter six are probably haploid sets of closely linked genes.     

The role of salinity and sodicity in the dieback of Acacia xanthophloea in Ngorongoro Caldera, Tanzania

Dieback of Acacia xanthophloea (Benth.) has opened up the once densely forested Lerai area in Ngorongoro Caldera, Tanzania. Soil samples were taken from profiles in the Ngorongoro Conservation Area and Lake Manyara National Park at sites of dieback and at sites with healthy A. xanthophloea trees. Dieback sites had significantly greater electrical conductivity (EC), water‐soluble Na⁺, K⁺, Cl^?, SO and sodium adsorption ratios (SAR) than healthy sites. The following mean values were recorded: EC (179 versus 70 mS m^?1; P < 0.001, Student's t‐test, n = 8 and 10, respectively; 40–60 cm); Na⁺ (99 versus 30 mmol_c kg^?1, P < 0.001, n = 7 and 8 respectively); K⁺ (11 versus 3 mmol_c kg^?1, P < 0.05); Cl^? (36 versus 7 mmol_c kg^?1, P < 0.01); SO (31 versus 5 mmol_c kg^?1, P < 0.01); and SAR (28 versus 8 mmol l^?1/2, P < 0.01). Water‐soluble Na⁺, Cl^? and SO concentrations in the Lerai profiles have probably resulted in toxicity and osmotic stress which contributed to dieback. Accumulation of salts may have occurred because of reduced flow of freshwater through Lerai and/or flow of water from Lake Magadi into Lerai. Forest recovery may be possible if salinity is reduced. Management strategies for reducing salinity have been implemented and included re‐establishing streams that flow through Lerai. Exclusion of elephants (Loxodonta africana) from Lerai is another management strategy presently under consideration.