Use of wheat bran as a nutritive supplement for the production of ethanol by Zymomonas mobilis

A strain of Zymomonas mobilis (ZYM-TS 1) was isolated from fermenting palm wine (toddy). In addition to glucose, sucrose, and fructose, the organism utilized hydrolysates of corn ( Zea mays ) flour, corn starch and ragi ( Eleusine coracana ) flour. Amounts of ethanol produced in media (adjusted to 10% (w/v) total carbohydrates) fortified with wheat bran extract only, were comparable with those obtained from the defined media containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, and MgSO₄.7H₂O. The results indicate that wheat bran extract can supply all the necessary nutrients required for ethanol production by Zymomonas mobilis .     

Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation

Abstract The effects of combinations of desiccation and exposure to O₂ were studied in pure cultures of Methanosarcina barkeri strain Fusaro and in a new Methanosarcina strain and a new Methanobacterium strain which were both isolated from dry oxic paddy soil. Incubation of bacterial suspensions under air for 200 min resulted in a decreased potential to produce CH₄, but not in a decreased viability. The inhibitory effect of O₂ slightly increased with increased salt concentration. Desiccation of bacterial suspensions under N₂ resulted in reduction of viability to 10% and of potential CH₄ production to 0.6%. Desiccation of bacterial suspensions under air resulted in a larger decrease of both viability (0.5%) and potential CH₄ production (0.03%). This decrease was smaller at rapid compared to slow desiccation. Survival and potential CH₄ production were further inhibited when the suspension was dried in the presence of sand grains or glass beads coated with FeS or FeNH₄PO₄. However, survival and potential CH₄ production increased dramatically in the presence of pyrite (FeS₂) grains. Then, as much as 10% of the initial methanogenic population survived oxic desiccation. This relatively good resistance is in agreement with observations that methanogens in rice fields survive the periods when the paddy soil is dry and oxic.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium

The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape ( Orobanche minor Sm.), a root holoparasite, by its host red clover ( Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K₂SO₄) concentrations up to 100 mg l^–1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH₂PO₄) was highly inhibitory even at concentrations as low as 1 mg l^–1 (based on P). Nitrate (NaNO₃) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l^–1 (based on N). Ammonium [(NH₄)₂SO₄] had no effect at 2 mg l^–1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l^–1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.     

The Glycine-Glomus-Bradyrhizobiun symbiosis. XI. Nodule gas exchange and efficiency as a function of soil and root water status in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants were inoculated with a HUP− strain of Bradyrhizobium japonicum (Nitragin 61A118) and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol & Gerd.) Gerd. and Trappe or fertilized with KH₂PO₄ (nonVAM). They were grown for 50 days in a growth chamber and harvested over a 4-day drought period during which available soil water decreased to 0. Nodule P concentrations and P-use efficiency declined linearly with soil and root water content during the harvest period in both VAM and nonVAM plants. Nitrogenase activity, estimated from H₂ evolution and C₂H₂ reduction data, was also a linear function of declining nodule P concentrations and CO₂-exchange rates and showed simular patterns in both treatments. Hydrogen evolution and the relative efficiency of N₂ fixation, on the other hand, reacted differently to increasing drought in VAM and nonVAM plants. Differences in the responses of nodule activity in VAM and nonVAM plants to drought are interpreted in terms of demand for nodule P and carbohydrates and of the effects of dehydration on O₂ diffusion through nodule tissue.     

The Glycine-Glomus-Bradyhizobium symbiosis. VIII. Phosphorususe efficiency of CO2 and N2 fixation in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants nodulated by Bradyhizobium japonicum strain USDA 110 were grown in pot cultures in severely P- and N-deficient soil and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or fertilized with a high (HP) or low (LP) level of KH₂PO₄ (0.6 or 0.3 m M , respectively), After 7 weeks of growth, nodule and chloroplast activities (C₂H₂ reduction and CO₂ exchange rate) were determined. Photosynthetic P-use efficiency of CO₂ fixation was significantly higher in VAM than in HP plants, while that of nitrogenase activity was lower. The LP plants were intermediate in both respects. The ratio of nodule to chloroplast activity [mol C₂H₂ reduced (mol CO₂ fixed)⁻¹] was highest in HP and lowest in VAM plants. Root colonization by the VAM fungus significantly increased nodule number and dry weight and reduced nodule specific mass and activity in comparison to HP plants. In spite of lower nodule activity, VAM plants were significantly larger and had higher N concentrations than the HP plants. The results suggest nonnutritional. VAM-elicited and host-mediated effects on the symbiotic functions of the legume association.     

Changes in abundance, composition and controls within the plankton of a fertilised arctic lake

1. An oligotrophic arctic lake was fertilised with inorganic nitrogen and phosphorus as (NH₄)₂ NO₃ and H₃PO₄ for five summers. The loading rate was 1.7–2.5 mmol N m^–2 day^–1 and 0.136–0.20 mmol P m^–2 day^–1 which is two to three times the annual loading of lakes in the area. The heterotrophic microzooplankton community was enumerated during the experiment as well as 1 year pre- and post-treatment.
2. The structure of the microplankton community changed from a nutrient limited system, dominated by oligotrich protozoans and small-particle feeding rotifers, to a system dominated by a succession of peritrich protozoans and predatory rotifers. These peritrich protozoans and predatory rotifers were not present prior to fertilisation and never constituted more than a small fraction of the biomass in other lakes at the research site. The average biomass of the rotifers and protozoans was more than seven and a half times larger by the end of fertilisation than it was initially.
3. Because of the increases in numbers of individuals in these new taxa, the structure of the microbial food web changed. When fertilisation stopped, most parameters returned to prefertilisation levels within 1 year.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Isolation and characterization of α-aminoadipate-δ-semialdehyde overproducing mutants from yeasts

Abstract We isolated from Candida maltosa mutants lacking saccharopine reductase ( lys9 ) and saccharopine dehydrogenase ( lys1 ). They accumulated α-aminoadipate-δ-semialdehyde (AASA) in the cell and excreted it into the culture medium. In the presence of 15 g glucose/l, 1.25 g NH₄H₂PO₄/l and 50 mg l -lysine/l in a minimal salt medium C. maltosa G285 ( lys1 ) produced about 80–90 mg AASA/l within 48 h. It is the first report of lysine-requiring yeast mutants that accumulate and excrete AASA. In contrast, Pichia guilliermondii lys9 mutants lacked this AASA overproduction. The AASA accumulation by C. maltosa mutants may be explained by the low feedback regulation of their homocitrate synthase and the equilibrium of the enzyme reactions involved in the lysine biosynthesis.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

Methyl sulfides as intermediates in the anaerobic oxidation of methane

While it is clear that microbial consortia containing Archaea and sulfate-reducing bacteria (SRB) can mediate the anaerobic oxidation of methane (AOM), the interplay between these microorganisms remains unknown. The leading explanation of the AOM metabolism is 'reverse methanogenesis' by which a methanogenesis substrate is produced and transferred between species. Conceptually, the reversal of methanogenesis requires low H₂ concentrations for energetic favourability. We used ¹³C-labelled CH₄ as a tracer to test the effects of elevated H₂ pressures on incubations of active AOM sediments from both the Eel River basin and Hydrate Ridge. In the presence of H₂, we observed a minimal reduction in the rate of CH₄ oxidation, and conclude H₂ does not play an interspecies role in AOM. Based on these results, as well as previous work, we propose a new model for substrate transfer in AOM. In this model, methyl sulfides produced by the Archaea from both CH₄ oxidation and CO₂ reduction are transferred to the SRB. Metabolically, CH₄ oxidation provides electrons for the energy-yielding reduction of CO₂ to a methyl group ('methylogenesis'). Methylogenesis is a dominantly reductive pathway utilizing most methanogenesis enzymes in their forward direction. Incubations of seep sediments demonstrate, as would be expected from this model, that methanethiol inhibits AOM and that CO can be substituted for CH₄ as the electron donor for methylogenesis.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Mice Deficient in Group IV Cytosolic Phospholipase A2 Are Resistant to MPTP Neurotoxicity

Abstract: Phospholipase A₂ (PLA₂) enzymes are critical regulators of prostaglandin and leukotriene synthesis, and they may also play an important role in the generation of intracellular free radicals. The group IV cytosolic form of phospholipase A₂ (cPLA₂) is regulated by changes in intracellular calcium concentration, and the enzyme preferentially acts to release arachidonic acid esterified at the sn -2 position of phospholipids. We examined the susceptibility of mice carrying a targeted mutation of the cPLA₂ gene to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Mutant mice have no functional cPLA₂ activity. Mice that were homozygous for the mutation (cPLA₂^−/−) were significantly resistant to MPTP-induced dopamine depletion as compared with littermate control (cPLA₂^+/+) and heterozygous mice (cPLA₂^+/−). These findings provide evidence that cPLA₂ plays a role in MPTP neurotoxicity and suggest that cPLA₂ may play a role in the development of Parkinson's disease in humans.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum

Eukaryotic cells contain a variety of cytoplasmic Ca²⁺-dependent and Ca²⁺-independent phospholipase A₂s (PLA₂s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca²⁺-independent PLA₂ (iPLA₂) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA₂ antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans -Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA₂ enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events.     

Properties of two pectin lyases produced by Aureobasidium pullulans LV 10

Two pectic lyases, L₁ and L₂, from culture liquids of Aureobasidium pullulans LV 10 were partially purified by ultrafiltration, CM-Sepharose 6B, DEAE-cellulose and/or Sephadex G 100 column chromatography, and characterized. L₁ and L₂ showed optimum activity at pH 5 and 7.5 respectively, and at 40°C. The molecular weights of the enzymes determined by gel filtration were estimated to be 89000 1000 and 55000 1000 for L₁ and L₂ respectively. Both lyases were activated by Ca²⁺ ions. L₁ attacked highly esterified pectins, L₂ attacked low methoxy-pectins in preference to polygalacturonic acid.