Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Effects of sucrose on blood avidity in mosquitoes.

Within 10 min after engorging on 10% sucrose, most females of Aedes aegypti do not seek a human blood meal, but remain quiescent and unresponsive to a human hand in a 1 ft³ cage. The duration of this inhibition in blood avidity varies greatly among individuals, but may last for 2 to 5 hr after drinking sucrose. There was no specific correlation between abdominal distension and blood avidity. When females engorged on varying concentrations of sucrose, it was found that as the concentration increased, fewer mosquitoes would take blood when it was offered after 1 hr. Only 25% of the females which engorged on 0·5 to 1 M sucrose took human blood at this time. As the concentration of sucrose increases, there is a marked decrease in spontaneous flight activity during the first hour after feeding. When unfed females are injected with 10% sucrose or trehalose, none of them took blood for 3 hr, whereas 50% of the saline-injected controls fed on blood.     

Adenosine Triphosphate Acceleration of the Nephridial Apparatus of Paramecium multimicronucleatum*

SYNOPSIS. Paramecium multimicronucleatum was exposed to various external concentrations of adenosine triphosphate (Na₂ATP) to determine the effects thereof on the cycling rate of the nephridial apparatus. Normal rate was found to vary from 3.46 to 4.28 cycles/min with a mean rate of 3.85 cycles/min at 20 C. Concentrations of ATP of less than 5 × 10⁻⁴ M caused only slight, very temporary acceleration of the cycling rate. At 5 × 10⁻⁴ M the cycling rate was accelerated less than 15%. At 3 × 10⁻³ M cycling rate was accelerated, varying from 5.35 to 7.24 cycles/min, with a mean accelerated rate of 6.25 cycles/min, a mean aoceleration of 88.3%. Changes in rate after addition of 5 × 10⁻³ M ATP ranged from a decrease of 6.2% to an increase of 1.8%, with the nephridial apparatus ultimately stopping. At higher concentrations, stoppage was almost immediate. Paramecium is rapidly dehydrated by the ATP-accelerated cycling of its nephridial apparatuses, with a net loss of 27% of its volume in 6 minutes in the 3 × 10⁻³ M ATP.     

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> on <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick larvae

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT₅₀ values obtained were 134.6, 27.2 and 24.8 days for concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT₅₀ values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 10⁶, 10⁷ and 10⁸ conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.     

Respiratory functions involved in the induction of puffs in Drosophila salivary glands

Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various substances affecting oxidative phosphorylation. After an incubation time of 1–3 h changes in puff size and in cellular ATP level were registered. 10^?6 M trinactin, 10^?5 or 10^?4 M oligomycin both induce puff 63BC together with some other puffs and reduce the cellular ATP level by about 80–90%. The trinactin-dependent puff induction can be inhibited, if the medium is supplemented with 10^?3 M ATP or 10^?3 M ITP or 10^?6 M antimycin or 10^?2 M KCN. The effect of exogenous ATP is prevented by adding 10^?6 M oligomycin to the incubation mixture; 10^?6 M oligomycin alone, however, has no inductive effect on 63BC. The presence of exogenous ITP, furthermore, prevents the ATP level from being reduced by trinactin. 10^?4 M atractyloside lowers the ATP level by about 75 %, whereas a puff induction cannot be observed. The same is true for various concentrations of KCN. It is concluded that ATP itself is not involved directly in the regulation of puff activity but that it acts on a phosphorylating reaction that can be inhibited by oligomycin.     

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min⁻¹ · mg⁻¹, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K_m-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H⁺-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I₅₀ = 2 μM) or to SW 26 (I₅₀ = 0.5 μM) than that of the original membrane fractions.     

The effect of seed conditioning,short-term heat shock and salicylic,jasmonic acid or brasinolide on sunflower (Helianthus annuus L.) chilling resistance and polysome formation

The aim of this study was to develop the method for increasing resistance of sunflower seedlings ‘Wielkopolski’ to chilling. Seeds were conditioned at 25 °C for 2 days in water to 15, 20 and 25 % moisture content or in salicylic or jasmonic acid in concentration of 10^?2; 10^?3 and 10^?4 M or brassinolide in concentration of 10^?6; 10^?8 and 10^?10–15 % moisture content. After 2 days of incubation the conditioned seeds were heat shocked at 45 °C for 0, 30, 60, 120 and 240 min and 5 mm seedlings were exposed to chilling at 0 °C for 21 days. The effectiveness of the methods was assessed by evaluation of roots growth in Phytotoxkit Microbiotest, changes in the activity of dehydrogenases, the integrity of the cytoplasmic membrane and formation of polysomes after seedling were returned to 25 °C for 72 h. Seeds were conditioned at 25 °C for 2 days in water to 15 % moisture content and then heat shocked at 45 °C for 2 h decreased chilling injury of seedlings expressed by subsequent growth of the roots, electrolyte leakage, dehydrogenases activity and polysomes formation. Application of heat shock of 45 °C for 2 h during seed conditioning additionally provided seedling protection against subsequent chilling conditions. Brasinolide, salicylic acid or jasmonic acid applied during seeds conditioning exhibited further beneficial effect on seedling resistance to chilling. The most pronounced effect was obtained due to seed conditioning to 15 % moisture content in solutions of brassinolide in concentration of 10^?8 M. After 2 days of imbibition treated in this way seeds were exposed to heat shock at 45 °C for 2 h. The role of physiological events in improvement of sunflower chilling tolerance are discussed.     

Semen backflow after insemination and its effect on fertilisation results in sows

The aim of the present study was to investigate the volume of and number of spermatozoa in semen backflow during and after insemination, and the effect of backflow on fertilisation results assessed at day 5 of pregnancy. Multiparous sows (n=140) were artificially inseminated with either (1, 3 or 6)×10⁹ mixed spermatozoa from three boars in a constant volume of 80 ml. Backflow of semen was measured three times: during insemination (M1); during the first half hour after insemination (M2); and from 0.5 h until about 2.5 h after insemination (M3). Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation. Sows were sacrificed at 120±0.4 h after ovulation to assess the results of fertilisation. Every sow had some backflow and the variation in volume, and number of spermatozoa within the backflow was high. The average semen backflow within 2.5 h after insemination was 70±3.4% of the volume and 25±1.4% of the spermatozoa of the inseminated dosage. The concentration of the backflow (% of the inseminated dosage) decreased with time after insemination from 65% at M1 to 40% and 26% at M2 and M3, respectively. The correlations between volume and number of spermatozoa were high: r=0.97, r=0.73 and r=0.81 in M1, M2 and M3, respectively. More than 5% of the inseminated spermatozoa in backflow during insemination affected fertilisation negatively in those sows inseminated with 1×10⁹ spermatozoa (P<0.05). Backflow after insemination had no effect on fertilisation results (P>0.05). Timing of insemination relative to ovulation and oestrus were not related to backflow during or after insemination (P>0.05). Of the sows which had backflow, those of parity 1 tended to have the highest proportion of sows with more than 5 ml backflow (47%; n=8 of 17) compared with sows from parity 2 and higher (24%; n=14 of 59) (P=0.075). It was concluded that excessive backflow of semen during insemination had a negative effect on fertilisation results when sows where inseminated with only 1×10⁹ spermatozoa. Causes of variation in backflow between sows were not clearly identifiable.     

Bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in clinical blood cultures

A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10^?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10^?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

Chromosomal aberrations induced by caffeine in somatic ganglia of Drosophila melanogaster

Nerve ganglia of third-instar larvae were treated with various doses of caffeine (5×10^?4, 10^?3, 5×10^?3, 10^?2 and 2×10^?2 M) for 2 h at 25±1°C. The ganglia were fixed at set time intervals after treatment so that the effect of caffeine in different stages of the cell cycle could be observed. Chromatid aberrations were induced only when the caffeine was administered in G₂ or approaching mitosis. No aberrations were observed after treatment in S or early G₂. In relation to the different doses administered, a threshold effect was evidenced, the number of aberrations increasing in a marked way at doses exceeding 5×10^?3 M. These data indicate, that the effect observed in Drosophila melanogaster is similar to that described by Kihlman in animals and plants treated with caffeine at temperatures below 30°C.Results obtained in non-cytological tests (non-disjunction, chromosome loss, lethal recessives, dominant lethals) have so far given incomplete indications as to the mutagenicity of caffeine in Drosophila. The results we have obtained with the cytological test seem to contribute to a better definition of the mutagenecity.     

The gelation kinetics of platelet extracts

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca²⁺ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca²⁺ concentration up to about 2 μM. At 4–15 μM free Ca²⁺, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca²⁺ concentrations. Trifluoperazine (50 μM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca²⁺ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K⁺, ATP, cytochalasin E and colchicine. K⁺ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelations, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 μM) and colchicine (1 mM) interfered with the gelation process.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

ATPase Activity of the Type IC Restriction-Modification SystemEcoR124II

We have investigated the ATPase activity of the type IC restriction-modification (R – M) systemEcoR124II. As with all type I R – M systemsEcoR 124II requires ATP hydrolysis to cut DNA. We determined theK_Mfor ATP to be 10⁻⁵to 10⁻⁴M. By measuring ATP hydrolysis under different conditions and by simultaneously monitoring DNA restriction, methylation and ATP hydrolysis we propose that the order of events during restriction is: (1) binding ofEcoR124II to a non-methylated recognition sequence, (2) start of DNA-dependent ATP hydrolysis which continues even after restriction is complete, (3) restriction of DNA, (4) methylation of the product. Non-cleavable DNA substrates, such as recognition site containing oligonucleotides, also support ATP hydrolysis. Methylation can also occur prior to ATP hydrolysis and prevent DNA degradation.     

The effect of tube closures and media on the enrichment of Campylobacter jejuni

Enrichment of C. jejuni in metal, Morton-style capped tubes gave no growth. Cotton wool or sponge rubber plugged tubes yielded enrichment of C. jejuni to 1.6 × 10³ cfu per ml from an inoculation of 0.18 cfu per ml in 10 ml medium.Enrichment of C. jejuni from egg melange in cotton plugged tubes and bottles showed that the ratio of egg melange to broth should not exceed 4:1 in bottles and 2:3 in tubes. When enriching from incubating fertile eggs infected by C. jejuni a decreasing quantitative and qualitative recovery was experienced with increasing time of egg incubation. Five enrichment broths and two selective plating media were compared in this experiment. The medium of Doyle and Roman (Appl. Environ. Microbiol. 43, 1343–1353 (1982)) and a routine enrichment broth plus rifampin (brucella broth containing per litre: 50 ml lysed horse blood, 10 mg rifampin, 5000 IU polymyxin B, 10 mg vancomycin, 5 mg trimethoprim lactate and the reductants of George et al. (J. Clin. Microbiol. 8, 36–41 (1978)) were superior to BNP broth. All media showed decreased enrichment of C. jejuni with increasing time of egg incubation, when growth was only of the order of 2 × 10² cfu per ml at day 15 of egg incubation. Rifampin was required to suppress contamination by Proteus species and Gram-positive cocci.     

Biochemical characterization of the plasma membrane Ca2+ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

• 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM).
• 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
• 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa.
• 4.4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles.
• 5.5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.

     

Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

Highly sensitive enzyme thermistor determination of ADP and ATP by multiple recycling enzyme systems

An enzyme thermistor method for the determination of ADP and/or ATP with signal amplification by recycling procedures is described. Pyruvate kinase (PK) and hexokinase (HK) coimmobilised on aminopropyl-controlled pore glass were applied in a column reactor. Addition of an excess of phosphoenolpyruvate (PEP) and glucose leads to cofactor recycling and production of glucose-6-phosphate and pyruvate. In presence of PEP an amplification of the sensitivity up to 30 times was reached as compared with the HK-catalysed reaction alone. An additional signal amplification was accomplished by recycling the pyruvate leaving the first enzyme reactor in a second reactor containing L-lactate dehydrogenase, lactate oxidase and catalase. In the presence of NADH an overall amplification of the sensitivity for ATP or ADP up to 1700 times was found. The limits of detection were 6 × 10⁻⁵ M cofactor without recycling at all, 2 × 10⁻⁶M with recycling in the kinase bienzyme reactor and 1 × 10⁻⁸M with the dual recycling system.