On-line estimation of dissolved methane concentration during methanotrophic fermentations

Knowledge of the aqueous phase methane concentration is critical to understanding and controlling process kinetics in methanotrophic bioreactors. Unfortunately since no dissolved methane probe is commercially available, this data must be obtained off-line by the time-consuming gas-liquid partition method. In this study we demonstrate how knowledge of the reactor's k(L)a for oxygen combined with gas phase methane analysis can be used to continuously estimate the aqueous phase concentration of dissolved methane. The on-line estimation was verified in two reactor systems with greatly different values of k (L)a. In both systems the measured and calculated dissolved methane concentrations were in good agreement although dissolved methane was underestimated in both cases. The utility of this methodology was demonstrated by revealing a possible metabolic bottleneck in the model system.     

Basidiomycete cryopreservation on perlite: evaluation of a new method

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.     

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys and Asp in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn²⁺, Pb²⁺ and Cd²⁺ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K⁶⁹³ and D⁷¹⁴, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P_i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K⁶⁹³ and D⁷¹⁴: the substitution K693N eliminates the Zn²⁺-stimulated ATPase activity completely, although significant Zn²⁺-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P_i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P_i. In conclusion, K⁶⁹³ and D⁷¹⁴ are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

A Review of the Genera Croconema Cobb, 1920 and Pseudochromadora Daday, 1899 (Nematoda, Desmodoroidea): New Species from the Coasts of Kenya and Australia

This article is a review of the subfamily Desmodorinae (Nematoda, Desmodoroidea) and two related genera within this subfamily, Croconema Cobb, 1920 and Pseudochromadora Daday, 1899 with keys to genus or species level, genus diagnoses and lists of valid species. An emended diagnosis of, and discussion on, Sibayinema Swart & Heyns, 1991, is presented. Three new species are described: Croconema floriani sp.n. from the coast of Kenya, Pseudochromadora galeata sp.n. and P. securis sp.n. from the coast of Australia.     

Intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus

Summary We have investigated the interaction of Gluconacetobacter diazotrophicus, a non-nodulating endophytic nitrogen-fixing bacterium isolated from the intercellular spaces of sugarcane, with Arabidopsis thaliana and the crop plants maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), oilseed rape (Brassica napus), tomato (Lycopersicon esculentum), and white clover (Trifolium repens). Using seedlings grown aseptically in sucrose-containing culture media, we have shown that inoculation with very low numbers of G. diazotrophicus results in extensive intracellular colonization of root meristems and progressive systemic intracellular root colonization. Light microscopic examination of thin sections of resin-embedded root tips of Arabidopsis and these crop plants inoculated with β-glucuronidase (GUS)-labeled and with NifH promoter-GUS-labeled G. diazotrophicus showed blue-stained G. diazotrophicus within the cytoplasm of root cells, indicating that intracellular conditions were suitable for nitrogenase gene expression. Electron microscopy confirmed that these bluestained intracellular G. diazotrophicus were within membrane-bounded vesicles. We discuss whether these novel inoculations with G. diazotrophicus are likely to enable non-nodular endosymbiotic nitrogen fixation and whether these inoculations can also provide a plant system to investigate the endosymbiotic theory of the origin of eukaryotic organelles.     

Toxicity of depleted uranium on isolated rat kidney mitochondria

Background

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.

Methods

Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.

Results

Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.

General significance

Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

The aim of this study was to investigate associations of two candidate gene SNPs of the endocannabinoid receptor type 1 gene (CNR1) with overweight, obesity and obesity-related traits in Chinese retired women. The study subjects were a subsample of the Taizhou Retiree Women Cohort, consisting of 2812 retired women aged 50-64 years recruited from Taizhou, Jiangsu, China. Neither rs2023239 nor rs806381 polymorphism was significantly associated with body mass index-defined overweight and obesity or waist-to-hip-ratio-defined obesity. For obesity-related traits, rs2023239 was significantly associated with glutamate pyruvate transaminase (GPT) (median, 18.00 vs 17.00 for TT and TC genotypes, respectively, P=0.043). The rs806381 also showed significant association with triglyceride (TG) (mean±SD, 1.46±0.20 vs 1.53±0.20 for GA and GG+AA genotypes, respectively, P=0.013) under the dominant genetic model. In conclusion, the rs2023239 and rs806381 polymorphisms of CNR1 were not associated with increased overweight and obesity risk. But the rs2023239 polymorphism was significantly associated with GPT, and the rs806381 polymorphism was significantly associated with TG.     

Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli

Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation.