Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

THE IRREVERSIBLE INHIBITION OF BRAIN L-GLUTAMATE-I-DECARBOXYLASE BY (2RS,3E)-2-METHYL-3,4-DIDEHYDROGLUTAMIC ACID

Abstract— Incubation of chick embryo brain l -glutamate-1-dccarboxylase (GAD, EC 4.1.1.15) with (2RS,3E)-2-methyl-3,4-didehydroglutamic acid (MDG), a substrate analog of l -glutamic acid, results in a time-dependent irreversible inhibition of the enzymic activity. In the presence of 2.0 ± 10^-3 m inhibitor the half-life for inactivation is 11.6min. The inhibitor is a substrate for GAD and requires turnover prior to inactivating the enzyme and is therefore another example of the k _cat class of inactivator. The measured K _l is 6.6 ± 10^-4 m and the k _cat for its turnover is 1.01 ± 10^-3 s-¹ at 37°C (pH 7.2). The inhibitor has no effect on the apoenzymc or the holoenzyme treated with 1.0 ± 10^-3 m hydrazinc. Both l -and d -glutamate, but not mercaptoethanol, reduce the rate of enzymie inactivation by the inhibitor. The exceedingly high specificity implicit in the design of this inhibitor should render it useful in studies designed to uncover the physiological role of GABA.     

Effect of titanium dioxide concentration on the survival of Pseudomonas stutzeri during irradiation with near ultraviolet light

Cells of Pseudomonas stutzeri in suspensions of TiO₂ ranging in concentration from 0.5 to 4.0 g l^-1 were irradiated with a blacklight blue u.v. source displaying peak emissivity at approximately 370 mm. Irradiation under these conditions is known to result in the generation of lethal free radicals. During irradiation the suspensions were agitated, using a specially modified laboratory shaker, to ensure efficient exposure of the TiO₂. A u.v. radiation dose of 175 kJ m^-2 resulted in cell fractional survival ranging from 5.5 times 10^-5, at the lowest TiO₂ concentration, to 1.0 times 10^-6, at the highest TiO₂ concentration. The advantages of contactors employing TiO₂ suspensions are briefly compared to immobilized TiO₂ systems.     

Inactivation of Escherichia coli by ultraviolet light and hydrogen peroxide in a thin film contactor

Apparatus for irradiating enclosed thin liquid films with ultraviolet (u.v.) light (Λ= 253.7 nm) in combination with hydrogen peroxide was used to inactivate Escherichia coli in water. Hydrogen peroxide concentrations of 2.5, 5.0 and 10.0 g/I were used and in each case synergistic inactivation was observed. At the highest concentration, a fractional survival of 1.3 times 10^-3 was obtained after 20 min; this was decreased to 3.1 times 10^-6 by simultaneous u.v. irradiation.     

Effect of 2-chloroethylphosphonic acid on capsule formation and alkaloid content in Papaver somniferum

Application of different concentrations of ethephon (2-chloroethylphosphonic acid) to Papaver somniferum L. at the times of stem elongation, bud, and capsule formation produced different effects. Ethephon (10^-2 M ) retarded growth of the plant and inhibited capsule formation during stem elongation, significantly reduced capsule size during the flowering period, but did not alter capsule development during capsule formation. When applied during the period of stem elongation, ethephon (10^-3 M and 10^-4 M ) reduced capsule size; alkaloid accumulation was reduced by ethephon at a concentration of 10^-3 M , but slightly increased by 10^-4 M . Ethephon (10^-3 M and 10^-4 M ) did not alter capsule development or alkaloid content significantly when applied during bud formation, but stimulated capsule size and alkaloid content when applied during capsule formation. Pretreating the plants with Ag⁺ (silver nitrate) did not reverse the ethephon effect. The results suggest that capsule maturation and alkaloid accumulation in P. somniferum are modified by ethylene, which is produced as a result of exogenous ethephon treatment.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

Partial purification and biochemical properties of acid and alkaline phosphatases from Myxococcus coralloides D

F. GONZÁLEZ, M.E. FÁREZ-VIDAL, J.M. ARIAS AND E. MONTOYA. 1994. Acid phosphatase and alkaline phosphatase from vegetative cells of Myxococcus coralloides D were purified by two chromatographic steps. The molecular weights were estimated by gel filtration and SDS-PAGE. Optimum pH, stability, optimum temperature and thermal inactivation studies were made for both enzymes. EDTA and other chelating agents inhibited alkaline but not acid activity. Mg²⁺ activated the alkaline phosphatase, while the acid phosphatase was inhibited by fluoride. Both enzymes degraded a number of phosphomonoesters, but were unable to hydrolyse either polyphosphates or cAMP. The K _m values of the acid and alkaline phosphatases for p -nitrophenylphosphate were 5.0 times 10^-3 mol ***l^-1 and 1.5 times 10^-3 mol l^-1, respectively.     

3,5-Dichlorophenoxyacetic acid as a growth retardant

Tomato seedlings, grown in the glasshouse, were sprayed with solutions of 3,5-dichlorophenoxyacetic acid as sodium salt at 2 times 10^-5, 4 times 10^-5 and 10^-4 M. The treated plants became dark-green, dwarfed, and compact. After 6–7 weeks normal growth was resumed. Measurements and analytical data on treated and control plants are presented.     

2,4-Dichlorophenoxyacetic acid inhibition of potassium transport in barley seedlings

Growth, potassium uptake and translocation as well as transpiration rates were measured in intact low-salt barley seedlings ( Hordeum vulgare L. cv. Union) in the presence of different 2,4-D concentrations at pH 6.5. Growth was only affected at 10^-3 M .
Above 10^-7 M 2,4-D both uptake by the roots and transport to the shoots were inhibited. The inhibition at 10^-5 M remained constant for at least 24 h. Furthermore inhibition of uptake was measurable within 1 h. Excised roots and roots of intact plants showed the same uptake pattern.
It is suggested that the observed effects were caused by 2,4-D-induced changes in uptake and translocation systems in the roots. Pre-treatment with 10^-5 M 2,4-D had no effect upon subsequent potassium uptake. Transpiration was reduced within 1 h in 10^-4 or 10^-3 M 2,4-D, probably due to changes in water transport or root permeability.     

Resistance of a cased caddis larva to accidental entry into the drift: the contribution of active and passive elements

SUMMARY. . 1. The resistance to passive entry into the drift of first to fifth instar larvae of Allogamus auricollis (Pictet, 1834), a case-bearing caddis-fly, was investigated in the laboratory using an artifical stream channel.
2. Dead larvae in their cases were exposed to different current speeds. When the heads of the larvae were directed towards the water flow (frontal position), the current necessary to wash larvae away ranged from 3 cm s^-l (first instars) to 21 cm s^-1 for fifth instars. When the larvae were at right angles to the current (lateral position), these speeds were 2 and 9cm s^-1, respectively. In terms of force (Newtons), this passive resistance to drift ranged from 0.3x10^-6 N (first instar, frontal position) to 307.0x10^-6 N (fifth instar, frontal position). The data obtained in the experiments were in good agreement with values calculated from hydrodynamic equations, using biometric parameters of the larvae.
3. Total resistance to drift was studied by exposing living larvae to different current speeds. The speed just sufficient to wash larvae away ranged from 13 cm s^-1 in the first instar to 27.9 cm s^-1 in the fifth instar (frontal position). In terms of force, the total resistance to drift varied between 5.3x10^-6 N (first instar) and 547.5x10^-6 N (last instar).
4. The difference between total and passive resistance to drift was defined as'active resistance to drift', and is due to the effectiveness of a larva's attachment to the substrate. It ranged from 3.5x10^-6 N (first instar) to 222.8X 10^-6 N (last instar).     

Neuroendocrine stimulation of uterine gland protein synthesis in the tsetse fly, Glossina morsitans

ABSTRACT. The uterine gland of the tsetse fly Glossina morsitans morsitans Westw. synthesizes a secretion which nourishes the developing larva in the uterus. Aqueous extracts of the brain have been shown to stimulate the synthesis of the protein and amino acid components of this secretion from L- [U-¹⁴C]leucine by uterine gland tubules in vivo and in vitro. A linear dose response relationship was demonstrated in vitro with extract concentrations ranging from 1 × 10^-4 to 1 × 10^-2 brains μl^-1. The maximum response, a > 300% increase in the rate of protein and amino acid synthesis, was achieved with as little as 1 × 10^-2 brains μl^-1 The concentration of active factor(s) in the brain declined during a single interlarval period coincident with the period of release of secretion associated with larval growth. The stimulatory activity in brain extracts was destroyed by proteolytic enzymes indicating that it is probably a protein or peptide. Results suggest that the active factor(s) is a hormone responsible for the stimulation of uterine gland protein synthesis essential for larval nutrition.     

Interaction of triacontanol with gibberellin in control of lettuce seed germination

Triacontanol at concentrations from 2.3 × 10^-9 M to 2.3 × 10^-7 M did not affect the germination of lettuce ( Lactuca sativa L., cv. Grand Rapids) seeds in darkness, stimulated by light at 25°C or by benzyladenine at 31°C. Stimulation of seed germination by gibberellin A₃ (10^-5 M ) was significantly inhibited by triacontanol; the most effective concentration was 4.6 × 10^-8 M. Pulse experiments demonstrated that triacontanol was ineffective when applied later than gibberellin, whereas an inverse sequence of treatment caused an inhibition comparable to that resulting from continuous treatment of seeds with both factors. Possible interaction of triacontanol with gibberellin receptor is discussed.     

Effects of cytosine arabinoside and daunorubicin on survival and cell cycle progression of chinese hamster ovary cells

Abstract. Kinetic and cytotoxic effects of cytosine arabinoside (Ara-C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony-forming assay, respectively. With Ara-C alone, increasing drug concentrations between 10^-7 M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10^-4 M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 times 10^-7 M. It decreased the rate of progression through S phase and caused cells to accumulate in G₂, except at the highest concentration (2 times 10^-5 M), at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara-C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non-lethal dose of Ara-C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug-free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by druginduced redistribution of cells within the cell cycle, but may have been related to the effects of the non-lethal drug on DNA synthesis rate.     

Sequestration of 125I-Labeled p Nerve Growth Factor by Embryonic Sensory Neurons

Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 × 10^-11 M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 × 10^-9M. These two receptors are not a result of negative cooperatively, but apparently are different receptors. At 22°C the rate of association is 1 × 10⁷ M^-1 S^-1 and the rates of dissociation are 6.5 × 10^-4 s^-1 (type I) and 3.2 × 10^-2 s^-1'(type II). After binding, a time-dependent process occurs that makes the NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.     

Yam mosaic, a new potyvirus infecting Dioscorea cayenensis in the Ivory Coast

The major disease affecting Dioscorea cayenensis in the Ivory Coast is caused by a virus which was transmitted by mechanical inoculation to some Dioscorea spp. and Nicotiana benthamiana . In extracts of D. cayenensis leaves infectivity was lost after 10 min at 60 C but not 55 C and after dilution to 10^-3 but not 10^-2. A purification procedure is described. The virus particles are flexuous filaments c . 785 nm long. The virus was transmitted by four aphid species in the non-persistent manner, and is serologically related to four African potyviruses. The name yam mosaic virus is proposed; the present cryptogram is: */*:*/6:E/E:S/Ve/Ap, potyvirus group.     

Effects of pollutants in fresh waters on European non-salmonid fish I: Non-metals

As part of a programme of acquiring data for preparing standards to safeguard European, nonsalmonid, freshwater fish from pollution, toxicity tests were carried out in hard, well-aerated water. Asymptotic median lethal concentrations (LC50s) of undissociated ammonia, cyanide, nitrite and phenol to one or more of three species were determined. The LC50s were as follows: to common carp 16mg1^-1 as NO₂–N; to perch 0.1 mg1^-1 as HCN; to roach 0.35 mg1^-1 as NH₃-N, 0-11mg1^-1 as HCN, 10.1 mg1^-1 asNO₂-Nand 10mg^-1 as phenol. In order to define these LC50s, exposure periods within the range 0.3 days (phenol) to 14 days (nitrite) were required. Comparisons are made with other data and the tentative water quality 'criteria' proposed by the European Inland Fisheries Advisory Commission.     

Effect of alcohols on the association of photosynthetic fructose-1,6-bisphosphatase to thylakoid membranes

Aliphatic alcohols have a positive effect on the assoociation of pea ( Pisum sativum L. cv. Lincoln) chloroplast fructose- 1,6-bisphosphatase (FBPase; EC 3.1.3.11) with thylakoid membranes. The alcohol concentration needed to obtain a fixed percentage of enzyme association decreased with increased length of the aliphatic chain of the alcohol; maximum binding was obtained when the lysis medium contained, in molar fractions (or v/v percentages): 48×10^-4(T4 (2.4%), 26×10^-3 (10%), 40×10^-3 (15%), 76×10^-3 (21%), and 13×10^-2 (24%), of 1-butanol, 1-propanol, 2-propanol, ethanol, and methanol, respectively. A good correlation of binding with the octanol/water partition coefficient was observed. Since this coefficient constitutes a measure of hydrophobicity, we suggest that the binding of FBPase to the membranes occurs via hydrophobic clusters of both components.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

COMPARISON OF LUMINOL CHEMILUMINESCENCE WITH ATP BIOLUMINESCENCE FOR THE ESTIMATION OF TOTAL BACTERIAL LOAD IN PURE CULTURES

A rapid chemiluminescent assay of total bacterial load that is based on the oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as catalyzed by bacterial iron protoporphyrins is described and compared to the ATP bioluminescent assay of microbial biomass. An assay format that elicits linear light output response to a range of analyte concentrations of model compounds such as hematin and various heme-containing enzymes within the dynamic range of a BioOrbit 1251 luminometer is presented. When the assay was applied to eight pure bacterial cultures, the sensitivity was typically in the range of 10⁴-10⁵ cfu/ml, and was comparable to that obtained by the ATP assay. Similar levels of sensitivity can be derived from estimates of average values of 2.8 × 10^-18 mole of heme/cfu and 1 × 10^-19 mole of ATP/cfu. The potential of the luminal assay as an alternative rapid test for the estimation of total bacterial count in food and environmental samples is discussed.