The fermentation of glycerol byClostridium butyricum LMG 1212t2 and 1213t1 andC. pasteurianum LMG 3285

Summary In batch culture on reiinforced clostridial medium strain-dependent product profiles from glycerol revealed unusual fermentation products such as propionate and n-propanol with Clostridium butyricum LMG 1213t₁, and 1,3-propanediol with C. butyricum LMG 1212t₂ and C. pasteurianium LMG 3285. Only the latter two strains were able to grow on glycerol in a minimal medium. Nicotinamide adenine dinucleotide (NAD⁺)-dependent dehydrogenase activities were detected with 1,3-propanediol and n-butanol as substrate (the latter only after a lag period) in cell-free extracts of C. butyricum LMG 1212t₂ and with 1,3-propanediol, n-butanol and ethanol in cell-free extracts of C. pasteurianum LMG 3285. The data indicated the existance of a specific 1,3-propanediol dehydrogenase in both organisms. In a chemostat, C. butyricum LMG 1212t₂ converted 65% of the glycerol supplied as sole carbon and energy source to 1,3-propanediol without H₂ production. Increasing concentration of acetate in the inflow medium resulted in less 1,3-propanediol and more butyrate and H₂ production. C. pasteurianum LMG 3285 converted somewhat more than half of the glycerol supplied as sole energy and carbon source to n-butanol with significant concomitant H₂ production. This fermentation pattern was hardly affected by acetate as co-substrate. Offprint requests to: P. De Vos     

Genetic relationships among three chlorophyll-deficient mutants in peanut,Arachis hypogaea L.

Summary The genetic relationships of three chlorophyll-deficient mutant peanuts, lutescens (lu), aureus (au), and virescent (v) were studied under field and greenhouse conditions. The F₁ plants from crosses between these mutants produced phenotypically normal green. In F₂, aureus X virescent segregated 675 normal green : 225 virescent : 45 aureus : 15 virescent aureus : 64 seedling lethal, and lutescens X virescent segregated 45 normal green : 15 virescent : 3 lutescens : 1 seedling lethal. (Lutescens peanuts were seedling lethal in the field.) As previously reported, the F₂ of aureus X lutescens gave 225 normal green : 15 aureus :15 lutescens : 1 seedling lethal. The three chlorophyll-deficient factors (au, lu, and v) show independent inheritance. The recessive combinations from the parental types between aureus and virescent and between aureus and lutescens would produce plants with a combination of their respective parental characteristics, but the recessive combination between lutescens and virescent was nearly albino. The v-au and lu-au seedlings have a longer life span than the v-lu seedling has. The genotypes for the three mutants are tentatively identified as lutescens VV Au ₁ Au ₁ Au ₂ Au ₂ lu ₁ lu ₁ lu ₂ lu ₂ L ₁ L ₁ L ₂ L ₂, aureus VV au₁au₁ au₂au₂ Lu₁Lu₁ Lu₂Lu₂ L₁L₁ l₂l₂, and virescent vv Au₁Au₁ Au₂Au₂ lu₁lu₁ Lu₂Lu₂ l₁l₁ L₂L₂.     

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a ¹O₂-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by ¹O₂ but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether ¹O₂ was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the ¹O₂-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that ¹O₂-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Aiswarya Baruah and Klára Šimková contributed equally to the article.     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

Simultaneous determination of malachite green and its metabolite leucomalachite green in eel plasma using post-column oxidation

A rapid HPLC method with solid-phase extraction (SPE) clean-up for malachite green (MG) and leucomalachite green (LMG) in eel plasma was developed. MG and LMG were extracted with a buffered methanolic solution. The extract was subjected to aromatic sulphonic acid SPE. MG and LMG were eluted from the SPE column with methanol after a treatment with ammonia gas. The reconstituted eluate was analyzed on a Chromspher B column with acetonitrile-ion-pair buffer (ph 4.0) (6:4, v/v) as the mobile phase and detection at 610 nm after post column oxidation with PbO₂. The average recoveries for MG and LMG over the linear range of applicability (20–2500 ng/ml) were 82±1% and 83±1%, respectively. The limits of quantification were 5.0 μg/1 for MG and 0.9 μ/1 for LMG.     

Strain improvement of the xylose-fermenting yeastPachysolen tannophilus by hybridisation of two mutant strains

Two previously reported mutants ofPachysolen tannophilus, which accumulate ethanol more rapidly and in greater yield than the wild-type NRRL Y2460, have been cross-mated. Aneth 2-1 mutant which is unable to grow on ethanol, was mated with the mutant NO₃–NO₃-4 which possesses increased levels of pentose phosphate pathway enzymes. The new hybrid strain combines the properties of both parents and possesses improved characteristics for xylose fermentation to ethanol.     

Genetic variation in catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (<Emphasis Type="Italic">COMT</Emphasis>) and obesity in the prostate,lung, colorectal,and ovarian (PLCO) cancer screening trial

Catechol-O-methyltransferase (COMT) is an important modulator in the catabolism of extraneural dopamine, which plays an important role in drug reward mechanisms. It is hypothesized that genetic variations in the COMT gene, which can result in a three to fourfold difference in COMT enzyme activity, may be associated with several reward-motivated behaviors. The aim of our study was to examine the relationship between COMT polymorphisms with smoking, obesity and alcohol. Three single nucleotide polymorphisms (SNPs) in COMT were genotyped in 2,371 participants selected randomly from the screening arm of the PLCO Cancer Screening Trial after stratifying by sex, age, and smoking status. Smoking, obesity, and alcohol consumption were assessed by questionnaire. SNP and haplotype associations were estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from conditional logistic regression models, adjusted for race/ethnicity. The COMT Ex4-76C > G (Leu136Leu) polymorphism was statistically significantly associated with individuals who had >30% increases in BMI from ages 20 to 50 years, compared to those with 0–5% increase in BMI (0–5%) over the same age period: (CC is referent; OR_CG = 1.42, OR_GG = 1.46, P _trend = 0.06). By sex, the increased risk was further pronounced among females (OR_CG = 1.50, OR_GG = 2.10, P _trend = 0.03). Consistent with our analyses of single polymorphisms, individuals whose BMI increased >30% from ages 20 to 50 years were more likely than individuals with 0–5% increases in BMI to possess COMT haplotypes [COMT Ex3-104C > T–COMT Ex4-76 C > G–COMT Ex4-12 A > G] that included the variant allele for COMT Ex4-76 C > G: C-G-G (T-C-A is referent: OR_C-G-G = 1.33, 95% CI 1.01–1.77) and C-G-A (OR_C-G-A = 1.79, 95% CI 0.72–4.49). We observed no association between any of the COMT polymorphisms with smoking behavior or alcohol intake. The COMT Ex4-76C > G (Leu136Leu) polymorphism appears to play a role in large increases in BMI. The null association with smoking and alcohol and the pronounced association with increasing BMI among women further implicates COMT’s role in estrogen metabolism as a potentially culpable pathway. Our results support a need for comprehensive evaluation of COMT variations and their functional relevance as COMT may be an important molecular target to evaluate for new treatments regarding obesity.     

A Genetic Insight Into Peptide and Amino-Acid Utilization by Propionibacterium freudenreichii LMG 16415

In this note the genetic characterization of the peptide degrading system of Propionibacterium freudenreichii was addressed. Genomic fragments of P. freudenreichii subsp. freudenreichii LMG 16415 were cloned in Escherichia coli XL1 Blue, and those leading to an increase in peptidase-like activity using chromogenic substrates aminoacyl-β-naphtylamides (aminoacyl-βNA) were isolated and sequenced. This strategy allowed the identification of partial gene regions of P. freudenreichii LMG 16415 with significant similarity to proteins directly or indirectly involved in peptide and amino acid metabolism, i.e., an oligopeptide transporter, a D-amino acid oxidase, a muropeptidase, and an ABC transporter involved in osmoregulation similar to glycine betaine transporters.     

Influence of initial respiratory status on the short-and long-term activity of the trout red blood cell β-adrenergic Na+/H+ exchanger

The effects of ambient O₂ partial pressure and CO₂ partial pressure on the intensity of rainbow trout (Oncorhynchus mykiss) red blood cell -adrenergic Na⁺/H⁺ exchange were investigated. This was accomplished in vitro by continuously monitoring whole blood extracellular pH, partial pressures of O₂ and CO₂ and by measuring red blood cell water content and Na⁺ concentration before and 30 min after the addition of a catecholamine mixture (final nominal concentrations: 250 nmol·l^-1 adrenaline and 20 nmol·l^-1 noradrenaline). The experiments were performed under six different initial conditions combining two ambient partial pressures of CO₂ (1.50 and 6.75 torr) and three ambient partial pressures of O₂ (15, 30 and 150 torr). The activation of red blood cell Na⁺/H⁺ exchange (as indicated by marked reductions of whole blood pH) was followed by transient reductions in blood partial pressures of CO₂ and O₂ (2 min) resulting from the shift of the CO₂/HCO₃ ^- equilibrium within the cell and the subsequent binding of O₂ to the haemoglobin. The initial reduction in blood CO₂ partial pressure was followed by a rise reflecting the titration of plasma HCO₃ ^- by extruded H⁺. At low partial pressure of CO₂ (1.50 torr) there was a pronounced stimulatory effect of hypoxia on the initial intensity of the extracellular acidification (5 min), whereas at high CO₂ partial pressure (6.75 torr) hypoxia actually lowered the extent of the initial acidification. In all cases, Na⁺/H⁺ exchange activation was accompanied by increases in cell water content and red blood cell Na⁺ levles when measured 30 min after addition of catecholamines. Both hypercapnia and hypoxia increased the magnitude of these changes although the largest changes in cell water content and Na⁺ levels were observed under hypercapnic conditions. Thus, the long-term activity (as determined by measuring cell water and Na⁺ levels) of the Na⁺/H⁺ exchanger was enhanced both by hypercapnia and hypoxia regardless of the initial CO₂ partial pressure. The initial activity (5 min), on the other hand, although stimulated by hypercapnia was influenced by hypoxia in opposing directions depending upon the initial CO₂ partial pressure of the blood.Abbreviations RBC red blood cell(s) - Hb haemoglobin - pHe extracellular pH - P _bCO₂ blood partial pressure of CO₂ - P _bO₂ blood partial pressure of O₂     

Pathogenic Vibrio harveyi,in contrast to non‐pathogenic strains,intervenes with the p38 MAPK pathway to avoid an abalone haemocyte immune response

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD₅₀ (ORM4) = 2.5 × 10² CFU animal^?1). The two strains, LMG 4044T and LMG 7890 were non‐pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD₅₀ = 8.9 × 10⁴ and 1.6 × 10⁵ CFU animal^?1, respectively). To start unraveling the mechanism explaining these differences, the p38‐MAPK, a keyplayer in antimicrobial immune response, was studied. The non‐pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38‐specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response. J. Cell. Biochem. 106: 152–160, 2009. © 2008 Wiley‐Liss, Inc.     

Contractility of rat testicular seminiferous tubules in vitro: Prostaglandin F1α and indomethacin

The frequency of spontaneous in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dose-dependent manner by prostaglandin F_1α. PGF_1α treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGF_1α (10⁻⁷ M). Pre-injecting the rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. in vitro was more effective than pretreatment in vivo in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGF_1α restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the in vitro contractility of the tubules.     

Effects of growth conditions on the activities of superoxide dismutase and NADH-oxidase/NADH-peroxidase inStreptococcus lactis

An increase in the superoxide dismutase (SOD) activity inStreptococcus lactis was observed when the cells were grown at increased oxygen partial pressures or exposed to hyperbaric oxygen tensions. The NADH-oxidase/NADH-peroxidase activities inS. lactis increased in galactose-grown cells when cultivated in air compared with N₂/CO₂. This effect did not occur when glucose was the carbon source; however, an increase in the activities of these enzymes was observed in oxygen atmosphere. The correlation between SOD, NADH-oxidase/NADH-peroxidase, and the metabolic pathways involved is discussed. The effect of manganese on the SOD activity is also considered.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

Hemolymph oxygen content,oxyhemocyanin, protein levels and ammonia excretion in the shrimp Penaeus monodon exposed to ambient nitrite

Subadult Penaeus monodon (21.03±3.19 g) were exposed individually in sea water (30 mg·ml^-1) to 0.02 (control), 1.04, 5.02, 10.11 and 20.06 mg·l^-1 nitrite-N for 24h. Hemolymph pH, partial pressures of oxygen and carbon dioxide, bicarbonate concentration, oxyhemocyanin and protein levels, and whole animal ammonia-N excretion and nitrite-N uptake were determined. Ammonia-N excretion and hemolymph oxygen partial pressure increased, whereas hemolymph pH, HCO ₃ ^- , oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein levels decreased with increasing ambient nitrite-N. It is suggested that accumulated nitrite of P. monodon following exposure to ambient nitrite causes reduction of oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein in the hemolymph, and affects nitrogen metabolism and acid-base balance at low hemolymph pH.Abbreviations bw body weight - EC₅₀ concentration reducing growth rate by 50% that of controls - LC₅₀ median lethal concentration - nitrite-N nitrite concentration measured as nitrogen - PO₂ partial pressure of O₂ in hemolymph - PCO₂ partial pressure of CO₂ in hemolymph - sw sea water - ww wet weight     

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (K_s = 3.2 ± 1.6 μM, K_i = 38 ± 1.4 μM) and delF508 CFTR (K_s = 3 ± 1.8 μM, K_i = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (K_s = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (K_i = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5

In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17β-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August × Copenhagen Irish (ACI) strain. In a (BN × ACI)F₂ population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN × ACI)F₂ population.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the     

Response to oxygen of diazotrophic Azospirillum brasilense — Arthrobacter giacomelloi mixed batch culture

Azospirillum brasilense and Arthrobacter giacomelloi were grown together in batch culture under different oxygen pressures. The response to oxygen of growth, nitrogenase activity and respiration rate was determined. The two microorganisms were found to be able to coexist all over the range of partial oxygen pressures examined, that is from 0.004–0.20 bar. Nitrogenase activity by mixed culture of A. brasilense and A. giacomelloi always appeared higher than that of A. brasilense pure culture. Low respiratory activity at partial oxygen pressures higher than 0.02 bar by both pure and mixed cultures seemed not to account for the high nitrogenase activity and improved oxygen tolerance of the mixed culture.Abbreviations pO₂ partial oxygen pressure