Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

The evolution of saline lake waters: gradual and rapid biogeochemical pathways in the Basotu Lake District,Tanzania

The biogeochemical evolution of solutes markedly alters the chemistry in the closed-basin maar lakes that comprise the Basotu Lake District (Tanzania, East Africa). Examination of 11 (out of 13) lakes in the Basotu Lake District identified two distinct evolutionary pathways: a gradual path and a rapid path. During the course of biogeochemical evolution these waters follow either the gradual path alone or a combination of the gradual and rapid paths. Solute evolution along the gradual path is determined by all of the biogeochemical processes that for these waters appear to be tightly coupled to evaporative concentration (e.g. mineral precipitation, sorption and ion exchange, C0₂ degassing, and sulfate reduction). Rapid evolution occurs when mixing events suddenly permit H₂S to be lost to the atmosphere. The chemistry of waters undergoing rapid evolution is changed abruptly because loss of every equivalent of sulfide produces an equivalent permanent alkalinity.The Basotu Lake District in north central Tanzania is comprised of 13 maar lakes. They range in surface water conductivity from 592 to 24 000 µ S cm ^–1 (at 20°). Within these lake basins only a few of the variety of geo- and biogeochemical processes known to occur in lakes of this type are actually responsible for the gain and/or loss of individual solutes. For example, potassium appears to be taken up in the formation of illite. Calcium is precipitated as calcite. Magnesium interacts with alumino-silicate precursors to form a variety of clay minerals that contain magnesium (e.g. stevensite). This process is also known as reverse weathering. Sulfate is reduced to sulfide and subsequently lost as H₂S and/or metal sulfides. Alkalinity is lost owing to calcite precipitation and as a consequence of reverse weathering. Alkalinity is gained in the form of extra permanent alkalinity when sulfide is lost from these waters (via metal sulfide precipitation or gaseous emission to the atmosphere). Rapid (punctuated) evolution can occur in any lake containing anoxic waters providing that mixing events take place which cause H₂S to be lost to the atmosphere.Peter Kilham died on March 20, 1989, in Kisumu, Kenya, while working as part of a research team on Lake Victoria.     

Specific antibodies to bovine choline acetyltransferase raised in mice immunised with small amounts of partially purified enzyme

Choline acetyltransferase was purified approximately 18,000-fold from 300 g of bovine caudate nuclei to a specific activity of 21 μmol min mg protein. The overall procedure used was: extraction of the enzyme by high salt concentration, chromatography on carboxy-methyl-Sephadex, precipitation by ammonium sulphate, affinity chromatography on Blue-Sepharose and, finally absorption on hydroxylapatite. When the enzyme absorbed on hydroxylapatite was injected into mice, it provoked reproducibly a transient production of ‘inhibitory’ antibodies, followed by higher antibody titres mainly of ‘non-inhibitory’ type. These responses were elicited by injecting less than a total of 20 μg of immunogen. The highest antibody titre was obtained less than 2 months following the initial immunisation. Species cross reactivity was investigated. This procedure should prove to be of value in the production of monoclonal antibodies to choline acetyltransferase.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Influence of the surface potential on the Michaelis constant of membrane-bound enzymes: effect of membrane solubilization

We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with M_r 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.