Genetic mapping,distribution, and properties of an aconitase isozyme in Anopheles albimanus (Diptera:Culicidae)

Electrophoretic analysis of the developmental stages and tissues of Anopheles albimanus showed that qualitatively similar allozymes of aconitase (Acon-2) occur at all stages, and the enzyme is widespread in every larval and adult tissues. Relative heat stabilities of the allozymes were investigated by electrophoresis of heated aqueous extracts and by heating the enzyme in situ in acrylamide gels after electrophoretic separation in Tris-citrate and Tris-maleate buffer systems. The pupal aconitase in the crude extract is more stable to heat than the larval and adult enzyme. The presence of citrate ions in the gel increased the stability of aconitase to heat. Studies of substrate specificities indicated that cis-aconitic acid is the best substrate but citric acid can also serve as a substrate. Zymograms developed with isocitric acid as a substrate showed no aconitase electromorphs and produced only isocitrate dehydrogenase bands. Aconitase has a pH optimum of 8.0 and this enzyme is completely inhibited if treated in situ with ethylenediaminetetra-acetic acid (EDTA), p-chloromercuribenzoate (PCMB), and urea at concentrations higher than 5mm, 5×10^–5 m, and 2 m, respectively. Acon-2¹⁰⁰ and Acon-2¹⁰⁵ do not respond differently to the above treatments. Genetic crosses involving a holandric translocation, pericentric inversions, visible mutants, and allozyme markers were analyzed to map the aconitase (Acon-2) locus on the left arm of chromosome 3. The gene sequence (and map distances) on 3L is centromere—esterase-8 (Est-8)—2—esterase-4 (Est-4)—25—esterase-2 (Est-2)—9—Acon-2—5—phosphoglucomutase (Pgm)—7—esterase-6 (Est-6).     

Study of properties of structural subunits of IgM immunoglobulin obtained by reduction with 2-mercaptoethanol or by oxidative sulphitolysis

IgM was isolated from pig serum by isoelectric precipitation and gel filtration. Different methods of breaking down the disulphide bonds and of isolating subunits of the IgM molecule—oxidative sulphitolysis and reduction by 0.1m 2-mercaptoethanol in the absence of a disaggregating agent, oxidative sulphitolysis in the presence of 6m urea and reduction by 0.3m 2-mercaptoethanol in medium containing 6m and 8m urea—were compared. Degraded material was separated by gel filtration on Sephadex G-100 or G-200 in 0.05m formic acid with 6m or 8m urea. Oxidative sulphitolysis or reduction by 0.1m 2-mercaptoethanol without a disaggregating agent did not yield pure H andl chains. Oxidative sulphitolysis was the more effective. Oxidative sulphitolysis in 6m urea medium severely damaged the material. Reduction of IgM by 0.3m 2-mercaptoethanol in 6m or 8m urea also altered its immunochemical properties. The possible presence of light chains in the heavy chain fraction cannot likewise be excluded in this case. The results are in agreement with experiments showing that the molecular weight of the IgM heavy chain is greater than that of the IgG heavy chain.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Monitoring Cl<Superscript>−</Superscript> Movement in Single Cells Exposed to Hypotonic Solution

Self-referencing ion - selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1–3 μm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (V_N) measured in 100 and 10 mM NaCl (ΔV_N = 57 ± 2 mV), but was slightly greater than ideal when comparing 1 and 10 mm NaCl (ΔV_N = 70 ± 3 mV). The response was also linear in the presence of 1 mm glutamate, gluconate, or acetate, 10 μm tamoxifen, or 0.1, 1, or 10 mm HEPES at pH 7.0. The ISE was ∼3 orders of magnitude more selective for Cl⁻ over glutamate or gluconate but less than 2 orders of magnitude move selective for Cl⁻ over bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was ‘poisoned’ by 50 μm 5−nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 ± 5 mOsm) solution. The increase in efflux peaked 7–8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 μm tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.     

Optimized procedures for purification and solubilization of basic fibroblast growth factor inclusion bodies

The efficiency of purification of basic fibroblast growth factor (bFGF) inclusion bodies using EDTA and nonionic detergents was improved from 25 to 40% by shifting the pH from 8.5 to strong alkaline conditions (pH 9.5 – 10.5). Complete dissolution of bFGF inclusion bodies by guanidinium hydrochloride (> 3 m) was independent of pH and the presence of reducing agents. In contrast, solubilization of bFGF inclusion bodies by urea was pH-dependent and increased in efficiency (e.g. from 0 to 100%) by increasing the pH (from pH 5.0 to 10.5 at 9 m urea). The purification and solubilization procedures are efficient for inclusion body concentrations corresponding to 10 and 100 g per l dry cell weight, respectively.     

Ratio of biological and chemical oxidation during the aerobic elimination of sulphide by colourless sulphur bacteria

Summary Culture conditions were evaluated for their relevance to chemical and biological oxidation of sulphide in aqueous solution. With a mixed culture of colourless sulphur bacteria sulphide oxidation was investigated over a concentration range of 0.005 to 3.5 mm S^2–, a pH range from 1.0 to 9.0 and temperatures from 10 to 40° C. Biological sulphide oxidation quickly decreased when fermentation conditions were suboptimal and the proportion of chemical oxidation increased with high pH values and sulphide concentration and increasing temperature. The optimal conditions for biological activity were found to be 3 mm S^2–, pH 7.0, and a mesophilic temperature (40° C).Offprint requests to: C. Plas     

The effect of cations on reassociation of the components of the cellulosome cellulase complex synthesized by the bacterium Clostridium thermocellum

The cellulosome multienzyme complex was dissociated into 12–14 components when incubated at 30° C in a reaction mixture that was buffered at pH 5.0 and was 50 mm with respect to sodium dodecyl sulphate and 10 mm with respect to both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT). The dissociated components reassociated into a complex when dialysed against 20 mm TRIS/HCl buffer, pH 7.7, containing 2.5 mm DTT. When incubated in the presence of Ca²⁺ and DTT the reassociated complex had the same activity to hydrogen-bond-ordered cellulose as the undissociated cellulosome. However, when Ca²⁺ ions were incorporated into the TRIS/HCl-DTT dialysis medium the reconstituted complex had very little activity towards cellulose. Other divalent cations such as Mg²⁺ and Ba²⁺ had the same effect, but the monovalent cation Na⁺ resulted in a complex that was very active on crystalline cellulose. The results are interpreted as indicating that the divalent cations bind to one or more of the dissociated polypeptide components and induce changes in conformation that prevent their reassociation into a complex with activity towards crystalline cellulose. Correspondence to: T. M. Wood     

Volume-dependent Glutamate Permeation Depends on Transmembrane Ionic Strength and Extracellular Cl<Superscript>−</Superscript>

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl^– permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl^– on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl^– was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 mM) or low (50 mM) ionic strength solutions on both sides of the membrane, Cl^– was always more permeable than Glu. When a transmembrane ionic strength gradient (150 mM extracellular: 50 mM intracellular) was set to drive water into the cell, and in the presence of extracellular Cl^–, Glu became up to 16-fold more permeable than Cl^–. Replacement of extracellular Cl^– with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.     

Chemotaxis towards sugars inChlamydomonas reinhardtii

Chemotaxis ofChlamydomonas reinhardtii 137c cells towards maltose and sucrose was observed by capillary assay. The threshold concentration was approximately 10^–5 m for maltose and 10^–3 m for sucrose. The peak accumulation of cells occurred at 10^–3 m for maltose and 10^–2 m for sucrose. A selection procedure for chemotaxis mutants was developed. Mutant strain CHE-1 was not attracted by maltose; mutant strain CHE-2 was not attracted by sucrose. Addition of attractant fully inhibited photoaccumulation of cells. After a period of time the photoresponse of cells recovered. Under the conditions of our experiments the period of adaptation lasted 15–20 min in wild-type cells and 4–5 min in mutant cells on the given sugar. Glucose and acetate did not attract cells ofChlamydomonas. Added to the medium, these compounds had no effect on the photoaccumulation of cells.     

Chloride Channel Function in the Yeast TRK-Potassium Transporters

The TRK proteins—Trk1p and Trk2p— are the main agents responsible for “active” accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration—qualitatively as expected for H⁺ coupling to K⁺ uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K⁺ or H⁺, but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K⁺-dependent and can be enhanced by K⁺ starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K_0.5 at 46 μM [K⁺]_out and 6.8 mM [K⁺]_out, and saturating fluxes of ∼5 mM/min and ∼10 mM/min (referred to intracellular water). These numbers are compatible with the normal K⁺-transport properties of Trk1p and Trk2p, respectively.This revised version was published online in August 2005 with a corrected cover date.     

Degradation of polycyclic aromatic hydrocarbons by an immobilized mixed bacterial culture

The mixed bacterial culture MK1 was capable of degrading a wide spectrum of aromatic compounds both as free and as immobilized cells. By offering anthracene oil or a defined mixture of phenol, naphthalene, phenanthrene, anthracene and pyrene (in concentrations of 0.1–0.2 mm, respectively) as sources of carbon and energy, a specific degradation pattern correlating with the condensation degree was observed. Regarding the defined mixture of aromatic hydrocarbons, complete metabolism was reached for phenol (0.1 mm) after 1 day, for naphthalene (0.1 mm) after 2 days and for phenanthrene (0.1 mm) after 15 days of cultivation. The conversion of anthracene (0.1 mm) and pyrene (0.1 mm) resulted in minimal residual concentrations, analogous to fluoranthene and pyrene of the anthracene oil (0.1%). Maximal total degradation for the tricyclic compounds dibenzofurane, fluorene, dibenzothiophene, phenanthrene and anthracene of the anthracene oil (0.1%) occurred after 5 days. In general, a significant metabolisation of the tetracyclic aromatic hydrocarbons fluoranthene and pyrene was observed after the degradation of phenol, naphthalene and most of the tricyclic compounds. Doubling the start concentrations of the polycyclic aromatic hydrocarbons effected higher degradation rates. Cell growth occurred simultaneously with the conversion of phenol, naphthalene and the tricyclic compounds. The immobilized cells showed stable growth and, compared to freely suspended cells, the same degradation sequence as well as an equivalent degradation potential — even in a model soil system. Correspondence to: I. Wiesel     

6-Phosphogluconate dehydrogenase in cell free extracts of Escherichia coli K-12

Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10^-2 m MgCl₂ produced maximal activity, whereas higher concentrations caused inhibition. The K _m values were 2.5×10^-4 m for 6-phosphogluconate and 2.5×10^-5 m for NADP⁺ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl₂ buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Urea and thiourea transport in Aspergillus nidulans

Wild-type Aspergillus nidulans has an active transport system specific for urea which concentrates urea at least 50-fold relative to the extracellular concentration. It is substrate concentration dependent, with an apparent K _m of 3×10^–5 m for urea. Competition studies and the properties of mutants indicate that thiourea is taken up by the same system as urea. Thiourea is toxic at 5mm to wild-type cells of Aspergillus nidulans. Mutants, designated ureA1 to ureA16, resistant to thiourea have been isolated, and transport assays and growth tests show that they are specifically impaired in urea transport. The mutant ureA1 has a higher K _m value than the wild type for thiourea uptake. The ureA locus has been assigned to linkage group VIII. ureA1 is recessive for thiourea resistance while semidominant for the low uptake characteristic. The urea uptake system is under nitrogen regulation, with l-glutamine as the probable effector. The mutants, meaA8 and gdhA1, which are insensitive to ammonium control of many nitrogen-regulated metabolic systems, are also insensitive to ammonium control of urea uptake, but both are sensitive to l-glutamine regulation.Formerly at the Department of Genetics, University of Glasgow, Glasgow, Scotland.     

Relative efficiency of selection methods to improve a ratio of two traits in Tribolium

Summary Three generations of upward selection for the egg mass/adult weight ratio were carried out in Tribolium castaneum. The experiment involved five lines: E — selected for increased egg mass; W — selected for decreased adult weight; R — selected for egg mass/adult weight directly; L — linear index selected with economic weights of m ₂: -m ₁ egg mass to adult weight (m ₁ and m ₂ are the means for adult weight and egg mass, respectively); NL — nonlinear index selected. Adult weight (7 days after adult emergence) and egg mass (between 7 and 11 days) were measured. The NL, E, and L lines had the greatest observed responses for the ratio; the R and W lines were not effective in improving the egg mass/adult weight ratio. It was expected that the NL line would be superior to the E, L, and R lines, and that the W line would respond the least. Observed response was significant for egg mass in the NL line, and for adult weight in the E, W, and R lines. Strong selection to increase egg mass seems to represent the optimal criterion for the ratio to be improved. The usefulness of nonlinear indices as selection criteria to improve a nonlinear trait, previously found to be optimal for a trait defined as the product of two component traits, appears to hold also for the selective improvement of the ratio of two traits. Serious limitations expected for direct selection of the ratio have been confirmed in this experiment.     

Dissociation of yeast ribosomes; The effects of hydrostatic pressure and KCl

The dissociation of free ribosomes at elevated concentrations of KCl is dependent on hydrostatic pressure. The pressure necessary for the dissociation is determined for KCl concentrations ranging from 0.1–0.4 m. It varies between 425 kg cm^-2 at 0.1 m and 10 kg cm^-2 at 0.4 m. The partial dissociation of complex ribosomes in KCl is dependent on hydrostatic pressure in the same way as the complete dissociation of free ribosomes. Therefore, it is concluded that mRNA and peptidyl-tRNA do not contribute to the stability of the ribosome under these conditions.     

A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol

Summary 5-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8m. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.     

The ultrastructural immunocytochemical localization of superoxide dismutase in the amphibian urinary bladder: Effect of aldosterone

Summary Transmission electron microscopy and immunohistochemistry, the latter employing the avidin—biotin—peroxidase (ABC complex) technique, were utilized to localize copper—zinc superoxide dismutase (CuZn—SOD) enzyme activity in the epithelial cells of the toad urinary bladder mucosa. This scavenger enzyme catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. In unstimulated epithelial cells, enzyme activity was seen in the cytosol of granular, mitochondrial-rich and goblet cells. The basal cells were generally devoid of enzyme activity. In addition to the cytosol, SOD activity was also seen in association with the apical plasma membrane of the epithelial cells. In the presence of the steroid hormone aldosterone (10^–7 m, 30 min—6 h), CuZn—SOD activity was markedly increased along the luminal mucosal membrane of granular, mitochondrial-rich and goblet cells. This increase was seen as early as 30 min after the addition of hormone, and as long as 6 h after treatment. The cytosolic reaction was usually decreased or absent under these conditions. From the data presented, it appears that CuZn—SOD is involved in electrolyte (sodium) transport in the epithelial cells of the toad urinary bladder. The latter may involve hormone-induced alterations in luminal cell membrane structure and chemistry.To whom reprint requests should be addressed.     

Prebiotic synthesis of diaminopyrimidine and thiocytosine

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40–85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5m NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.     

Type II callus production and plant regeneration in tropical maize genotypes

A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally, four media combinations of 15 or 30 μm dicamba with or without 88 μm AgNO₃ were used to study the effect of dicamba and AgNO₃ on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 μm and when AgNO₃ was added to the medium. A synergistic effect between 88 μm AgNO₃ and 30 μm dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best tissue culture performance and plant regeneration capacity. Received: 5 October 1996 / Revision received: 21 April 1997 / Accepted: 9 May 1997