The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Image processing technique for measuring specific growth rates of Gibberella fujikuroi

Summary Two methods were compared for estimating of Gibberella fujikuroi grown with different proportions of glucose and starch. They were, =Ln2 (V_r/Le) on Petri dish (V_r= rate of tip extension and L_e= mean hyphal length) and, ₂=d (LnX)/dt in stirred fermenter. Values of ₁ and ₂ were in close agreement with each other.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

Nickel transport in Alcaligenes eutrophus

Transport of nickel ions was studied in Alcaligenes eutrophus. Two transport systems for nickel ions exist to satisfy the nickel demand for the lithotrophic hydrogen metabolism. A major nickel transport activity exhibited an apparent affinity constant (K _m) of 17 M nickel chloride. This activity was competitively inhibited by Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺. A minor nickel transport activity was determined in the presence of high (0.8 mM) magnesium. This activity was not inhibited by Zn²⁺ or Mn²⁺; its K _m was determined to be 0.34 M nickel chloride. These kinetics suggested a second transport system in A. eutrophus. The membrane potential of A. eutrophus was decreased upon the addition of ammonium ions leading to a decreased nickel transport. This inhibition could be reversed by fructose or by hydrogen indicating an energy dependent nickel transport. Protonophores inhibited the nickel transport. However, inhibitors of ATP synthase like dicyclohexylcabodimide or venturicidin had little or no effect on nickel transport. These data indicated that the transport was coupled to the proton motive force.     

Influence of phosphate and nitrate supply on root hair formation of rape,spinach and tomato plants

Summary Experiments with tomato, rape and spinach in nutrient solutions have shown that the formation of root hairs is strongly influenced by phosphate and nitrate supply. Decreasing the phosphate concentration of the nutrient solution from 100 to 2 M P resulted in an increase of root hair length from 0.1–0.2 to 0.7 mm of the three plant species. Root hair density also increased by a factor of 2–4 when the P concentration was lowered from 1000 to 2 M. The variation of these two root properties raised the root surface area by a factor of 2 or 3 compared to plants well supplied with P. Root hair length was closely related to the phosphate content of the root and shoot material. On the other hand, spinach plants grown in a split-root experiment produced root hairs in solutions of high P concentration (1000M P) if the major part of the total root system was exposed to low P concentration (2 M P). It is therefore concluded that the formation of root hairs does not depend on directly the P concentration at the root surface but on the P content of the plant.Similar experiments with nitrate also resulted in an increase in length and density of root hairs with the decrease of concentration below 1000 M. In this case marked differences between plant species occurred. At 2 M compared to 1000 M NO₃ root hair length of tomato increased by a factor of 2, of rape by a factor of 5 and of spinach by a factor of 9. Root hair length was correlated, but not very closely, to the total nitrogen content of the plants. It is concluded, that the influence of nutrient supply on the formation of root hairs is a mechanism for regulating the nutrient uptake of plants.Dedicated to Prof. Dr. E. Welte on the occasion of his 70th anniversary.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Zinc protects against arsenic-induced apoptosis in a neuronal cell line,measured by DEVD-caspase activity

Acute and chronic arsenic exposure results in toxicity in humans and causes many neurological and other manifestations. For the first time the present study reports that zinc decreases arsenic-induced apoptosis and also confirms a single report of apoptosis induced by arsenic in a neuronal cell line. Apoptosis measured by DEVD-caspase activity peaked between 10 M and 20 M of arsenic trioxide. Higher concentrations of arsenic up to 40 M caused increasing cell death with diminishing DEVD-caspase activity. The beneficial effect of zinc was proportional to its concentration with a significant decrease in arsenic-induced DEVD-caspase activity at 50 M and 75 M zinc (P<0.05). This finding may be of therapeutic benefit in people suffering from chronic exposure to arsenic from natural sources, a global problem especially relevant to millions of people on the Indian subcontinent.     

Temperature effects on the photosynthetic response of C3 plants to long-term CO2 enrichment

To assess the long-term effect of increased CO₂ and temperature on plants possessing the C₃ photosynthetic pathway, Chenopodium album plants were grown at one of three treatment conditions: (1) 23 °C mean day temperature and a mean ambient partial pressure of CO₂ equal to 350 bar; (2) 34 °C and 350 bar CO₂; and (3) 34 °C and 750 bar CO₂. No effect of the growth treatments was observed on the CO₂ reponse of photosynthesis, the temperature response of photosynthesis, the content of Ribulose-1,5-bisphosphate carboxylase (Rubisco), or the activity of whole chain electron transport when measurements were made under identical conditions. This indicated a lack of photosynthetic acclimation in C. album to the range of temperature and CO₂ used in the growth treatments. Plants from every treatment exhibited similar interactions between temperature and CO₂ on photosynthetic activity. At low CO₂ (< 300 bar), an increase in temperature from 25 to 35 °C was inhibitory for photosynthesis, while at elevated CO₂ (> 400 bar), the same increase in temperature enhanced photosynthesis by up to 40%. In turn, the stimulation of photosynthesis by CO₂ enrichment increased as temperature increased. Rubisco capacity was the primary limitation on photosynthetic activity at low CO₂ (195 bar). As a consequence, the temperature response of A was relatively flat, reflecting a low temperature response of Rubisco at CO₂ levels below its k_m for CO₂. At elevated CO₂ (750 bar), the temperature response of electron transport appeared to control the temperature dependency of photosynthesis above 18 °C. These results indicate that increasing CO₂ and temperature could substantially enhance the carbon gain potential in tropical and subtropical habitats, unless feedbacks at the whole plant or ecosystem level limit the long-term response of photosynthesis to an increase in CO₂ and temperature.Abbreviations A net CO₂ assimilation rate - C _a ambient partial pressure of CO₂ - C _i intercellular partial pressure of CO₂ - Rubisco Ribulose-1,5-bisphosphate carboxylase - VPD vapor pressure difference between leaf and air     

Effect of senescence and hormone treatment on the activity of a β-1,3-glucan hydrolase in Nicotiana glutinosa leaves

Summary A high level of activity of a -1,3-glucan hydrolase is present in leaves of Nicotiana glutinosa and the enzyme is also present in the roots, midribs, petioles and stems. By comparison, very low levels of -1,4-glucan hydrolase are found throughout the plant. The activity of the -1,3-glucan hydrolase in leaves aged on the plant was found to increase 14-fold during the course of leaf senescence and to reach a maximum in yellow-green leaves. Detached leaves and leaf discs floated on water in the dark showed similar patterns of change.The increase in -1,3-glucan hydrolase activity during senescence is apparently not due to the loss of an inhibitor from young green leaves or to the formation of an enzyme activator in yellow leaves. The enzyme in yellow leaves was electrophoretically indistinguishable from that in green leaves. The hydrolase is not firmly attached to the cell walls and is not present in the particulate fraction sedimenting at 105400xg for 60 min. Within the leaf cell it is therefore likely to be located either in the cytoplasm or in an easily disrupted structure such as a vacuole.The relationship of the hydrolase to leaf senescence was investigated by examining the effect of plant hormones on the changes in level of hydrolase, protein and chlorophyll in leaf discs during senescence. IAA (10 M) and GA₃ (50 M) did not alter the normal patterns of change, whilst Kin (50 M) delayed the loss of protein and chlorophyll and also delayed and decreased the rise in hydrolase activity. In contrast, ABA (190 M) which increased the rate of loss of protein and chlorophyll, also caused a decrease in the rate and extent of the rise in hydrolase.Possible functions of the hydrolase in the leaf are discussed.Abbreviations used throughout text CM-pachyman carboxymethyl pachyman - CM-cellulose carboxymethyl cellulose - BSA bovine serum albumin - ABA abscisic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - Kin kinetin     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

Whilst reliable estimates of chiasma frequencies can usually not be obtained, the probability (b) of a chromosome arm to be bound by at least one chiasma can often be determined. In the absence of interference this probability equals (1–e ^–2), where 2 is the average chiasma frequency of the chromosome arm and the average crossover frequency or map length. In the presence of interference is shown to retain its genetic meaning as an additive metric that may describe the chromosome arm or other distinctive chromosome segment in terms of genetic recombination. It is a form of potential map length, comparable to, but numerically different from the regular map length. It is termed provisionally crossing-over potential.A chromosome with armsm andn with crossing-over potentials and will form ring bivalents with a frequency (1–e ^–2).(1–e ^–2); open bivalents with a frequency (1–e ^–2).e ^–2+(1–e ^–2).e ^–2; univalent pairs with a frequencye ^–2.e ^–2. Estimates of these frequencies yield equations from which and may be solved. In rye (Secale cereale) their ratio (q) is approximately two and differs from the mitotic arm length ratio of 1.4, indicating localization of chiasmata in the long arms.Graphs are given to show how, with constantq, the relation between the probabilitiesb _m andb _n of the two arms being bound changes with changing averageb.Data are presented on chiasma frequencies in M I, and compared with the frequencies expected in the absence of interference to give an impression of the degree of interference. Apparent fusion of chiasmata simulates interference.     

Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (K_m=1.4 M) was competitively inhibited rolipram (K_i=1.2 M) whereas PDE V (K_m=0.83 M) was competitively inhibited by zaprinast in the molar range (K_i=0.12 M). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions. (Mol Cell Biochem140: 171–175, 1994)     

Observations on phase-locking within the response of primary muscle spindle afferents to pseudo-random stretch

In order to uncover encoder properties of primary muscle spindle afferent fibers, time coupling (phase-locking) of action potentials on cyclic muscle stretch was studied by means of pseudo-random noise. In cats Ia action potentials were recorded from dorsal root filaments and the gastrocnemius muscles of one hind leg were stretched. The stimulus time course was a determined sequence of randomly varying muscle length which could be applied repeatedly (sequence duration 0.6 or 20 s). The noise amplitude (standard deviation of displacements) was varied between 5 and 300 m, the upper cut-off frequency of noise f _c was varied between 20 and 100 Hz. The responses to the consecutive pseudo-random noise cycles were displayed as raster diagrams and cycle histograms. Phaselocking characterized the responses at all noise amplitudes outside the near threshold range (>10 m). The higher and f _c , the stronger was the phase-locking of impulses on the stretch. When and f _c were selected to achieve high mean stretch velocities of about 500 mm/s, phase-locking was as precise as 0.15 ms, measured as the variability of spike occurrences with respect to stretch. The rasters obtained with low noise amplitudes (<40 m) showed a loose phase-locking and this gave insight into underlying mechanisms: The elicitation of action potentials caused by dynamic stretch can be prevented by a post-spike depression of excitability. This disfacilitation was very effective in counteracting weak stretch components within the random sequence and less effective or even missing when relatively strong stretch components could force the spike elicitation. This led to the reestablishment of phase-locked patterns. The results were discussed in relation to the known encoder models.     

Photosynthetic inhibition after long-term exposure to elevated levels of atmospheric carbon dioxide

The effect of long-term exposure to elevated levels of CO₂ on biomass partitioning, net photosynthesis and starch metabolism was examined in cotton. Plants were grown under controlled conditions at 350, 675 and 1000 l l^-1 CO₂. Plants grown at 675 and 1000 l l^-1 had 72% and 115% more dry weight respectively than plants grown at 350 l l^-1. Increases in weight were partially due to corresponding increases in leaf starch. CO₂ enrichment also caused a decrease in chlorophyll concentration and a change in the chlorophyll a/b ratio. High CO₂ grown plants had lower photosynthetic capacity than 350 l l^-1 grown plants when measured at each CO₂ concentration. Reduced photosynthetic rates were correlated with high internal (non-stomatal) resistances and higher starch levels. It is suggested that carbohydrate accumulation causes a decline in photosynthesis by feedback inhibition and/or physical damage at the chloroplast level.Abbreviations Ci internal CO₂ concentration - Chl chlorophyll - DMSO dimethylsulfoxide - HSD honestly significant difference (procedure) - MCW methanolchloroform-water - Pi inorganic phosphate - S.E.M. standard error of mean     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Micropropagation and ecorestoration of Vanda spathulata,an exquisite orchid

Node explants collected from flowering plants of Vanda spathulata, an endemic and exquisite orchid of Peninsular India and Sri Lanka, were cultured in Mitra medium with combinations of 4.4–88.8 m 6-benzyl adenine (BA) and 0.0–114.2 m indole-3-acetic acid (IAA). Combinations of 44.4 m BA with 17.1 or 28.5 m IAA and 66.6 mM BA with 28.5 or 40.0 m IAA induced maximum formation of 12.6 and 12.1 shoots / node, respectively, in a 6-month period. Subcultured nodal explants produced maximum of 6.1 shoots at combinations of 22.2–44.4 m 21 BA and 5.7–28.5 m IAA. Rooting of shoots occurred in medium containing 75 g l^–1 banana pulp and 5.7 m IAA within 3–9 weeks. Plantlets of 2–5 cm length possessing two to five roots established easily in community pots at 80–90% rates without hardening. Community potted plants introduced into forest segments at Ponmudi and Palode in Southern Western Ghats of India established at a rate of 50–70%.     

Guanylyl Cyclase Participates in the Mechanism of Glutamatergic Modulation of Acetylcholine Non-Quantum Release in the Rat Neuromuscular Junction

It was shown that glutamate (10 M to 1 M) suppresses in a dose-dependent manner the non-quantum release of acetylcholine from rat motor nerve endings; the release intensity was estimated by the H effect. The action of glutamate was completely eliminated by the blockade of guanylyl cyclase by 1 M ODQ. An increase in the intracellular cGMP concentration by 1 M dibutyryl-cGMP reduced the H effect in a similar manner as glutamate did.     

Stimulation by subsynaptosomal fractions of transmitter efflux from plain synaptic vesicle fraction

The effects were investigated of purified subsynaptic fractions on the efflux of radioactivity from a plain synaptic vesicle fraction which had incorporated [³H]dopamine. About 50% of the radioactivity incorporated into the plain vesicles (120 g protein) was liberated on exposure to purified synaptic membranes (30 g protein). The synaptic membrane-dependent efflux appeared to depend on both adenosine triphosphate and divalent cations, especially Ca²⁺. Of the subcellular fractions used, the heavy microsomal fraction showed the same effects as the synaptic membrane fraction. Purified synaptic junctions exhibited the strongest stimulating effects: the efflux was 2 times greater than that observed with synaptic membranes. The stimulating effects of myelin were less than oneseventh of those of synaptic junctional fraction. These observations may indicate that the transmitters are liberated by interaction of vesicle membrane with synaptic membrane in the presence of ATP and divalent cations.Abbreviations EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol bis-(-aminoethylether)-N,N-tetraacetic acid - AMP-PNP adenyl imidodiphosphate