Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels

An extensive series of experiments has been performed to study the mobility of DNA fragments ranging in size from 2.0 to 48.5 kilobose pairs. By varying the agarose concentration in the gels and the electric field strength, three DNA electrophoresis regimes were clearly identified: the Ogston regime (small DNA fragments in large pores of agarose), the reptation regime without DNA chain stretching (small pores of agarose and weak electric fields), and the reptation regime with DNA chain stretching (small pores of agarose, strong electric fields, and large DNA fragments). Here we report on the experimental identification of these regimes and on the conditions governing the transition between each of them. The onset of reptation and of stretching of DNA chains in gel electrophoresis are described quantitatively for the first time, and a phase diagram for the dynamics of DNA during electrophoresis is presented.     

A comparative study of acquired amidase activity in Pseudomonas species

Pseudomonas putida PP3 carrying dehalogenases I and II and Pseudomonas aeruginosa PAU3 carrying dehalogenase I coded for by plasmid pUU2 were able to grow on 2-monochloropropionic acid (2MCPA). Neither strain utilized 2-chloropropionamide (2CPA) as a carbon or nitrogen source for growth. Mutations in both strains to 2Cpa+ phenotypes (designated P. putida PPW3 and P. aeruginosa PAU5, respectively) involved the expression of an acquired 2CPA-amidase activity. The amidase followed by dehalogenase reactions in these strains constituted a novel metabolic pathway for growth on 2CPA. P. putida PPW3 synthesized a constitutive amidase of molecular mass 59 kDa consisting of two identical subunits of 29 kDa. For those amides tested this acquired enzyme was most active against chlorinated aliphatic amides, although substrate affinities (Km) and maximum rates of activity (Vmax) were poor. P. aeruginosa PAU5 acquired a 2Cpa+ phenotype by overproducing the A-amidase normally used by this species to hydrolyse aliphatic amides. The A-amidase had only slight activity towards 2CPA. However, with constitutive synthesis the mutant grew on the chlorinated substrates. Chloroacetamide (CAA) was a toxic substrate analogue for these Pseudomonas strains. A strain resistant to CAA was isolated from P. aeruginosa PAU5 when exposed to 1-10 mM-CAA. This mutant, P. aeruginosa PAU6, synthesized an inducible A-amidase. CAA-resistance depended upon the simultaneous expression of CAA-inducible amidase and dehalogenase activities.     

The influence of fluid mixing upon respiratory patterns for extended growth of a methylotroph in an air-lift fermenter

The influence of methanol dispersion and fluid mixing upon respiratory patterns observed during unlimited fedbatch growth of the methylotrophic bacterium Methylophilus methylotrophus has been investigated. A concentric tube air-lift fermenter was employed for which the mixing and fluid circulation patterns have been well characterized. Respiratory quotients showed a marked dependence upon the position in the vessel at which methanol was injected, the volumetric rate of such methanol addition, the fluid circulation time, and the local mixing behavior; the latter two factors of which are both determined by the air throughput. Such variations are discussed on the basis of simple mixing concepts and observations of fluid dispersion.     

Relaxation studies on the interaction of hexamethonium with acetylcholine-receptor channels inAplysia neurons

The mode of action of the cholinergic antagonist hexamethonium on the excitatory responses of voltage-clamped Aplysia neurons to acetylcholine (ACh) has been examined by voltage- and concentration-jump relaxation analysis. At steady-state concentrations of ACh hyperpolarizing command steps induced inward current relaxations to a new steady-state level (Iss). The time constants of these inward relaxations, tau f, which approximate the mean single-channel lifetime, were increased both by increasing the membrane potential and by lowering the bath temperature (Q10 = 3) but were not affected by increasing the ACh concentration over the dose range employed. In the presence of hexamethonium hyperpolarizing command steps produced biphasic relaxations of the agonist-induced current. tau f was reduced in a voltage-dependent manner, the degree of reduction increasing with hyperpolarization. Slow, inverse relaxations were also triggered in the presence of hexamethonium. The time constant of this relaxation was reduced by increasing membrane potential and hexamethonium concentration. Both the estimated association (kf = 5 X 10(4) M-1 . sec-1) and the estimated dissociation (kb = 0.24-0.29 sec-1) rate constants derived from a three-state sequential model for block by hexamethonium were independent of the membrane potential. Similar rate constants were estimated from experiments with the concentration-jump technique, which were also independent of the membrane potential over the range -50 to -110 mV. It is suggested that the voltage-dependent actions of hexamethonium may originate either from an alteration of the channel opening and closing rate constants through an allosteric interaction with the ACh receptor, rather than through an influence of the transmembrane electric field on the rate of drug binding, or through a fast reaction which is rate-limited by voltage-independent diffusion.     

The deposition of bacterial cells from laminar flows onto solid surfaces

In a previous article, the authors proposed a simple model for the rate of removal of bacterial cells from solid surfaces by fluid shear. This Model has been extended to include the deposition of cells from a flowing suspension. The theory is compared to experimentally obtained data for the deposition of Bacillus cereus cells onto the surface of glass capillaries. The effect of a hydrophobic surface, siliconized glass, and the addition of an antifoam agent to the suspension is also investigated.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

A PROCEDURE FOR THE MEASUREMENT OF HEMICHOLINIUM-3 IN BRAIN TISSUE

Abstract— The natural fluorescence of hemicholinium-3 (HC-3) was used to provide a method for determining its concentration in brain tissue following intraventricular administration of the compound to rats and rabbits. When hemicholinium-3 was perfused through the cerebral ventricles of the rabbit, the highest concentration was found in the pons-medulla oblongata. The effect of a number of compounds on the uptake of hemicholinium-3 in the brain was studied in rats. Choline and tetramethylammonium significantly reduced the uptake of HC-3 from the cerebral ventricles but tetraethylammonium, physostigmine and atropine had no effect. The results are discussed in relation to the known effects of hemicholinium-3 in the central nervous system.     

Oxidized and reduced nicotinamide–adenine dinucleotide phosphate in tissue suspensions of rat liver

1. The concentrations of NADP and NADPH(2) in homogenates of rat liver (expressed as mug./g. wet wt. of tissue homogenized) were compared with values obtained from intact samples of liver taken from the same female rat. With 0.25m-sucrose alone as the suspending medium, or in combination with tris buffer or 0.01-0.1m-nicotinamide, considerable decreases in the sum of the NADP+NADPH(2) concentrations were occasionally observed during 30min. storage of homogenates at 0 degrees . However, addition of 0.5m-nicotinamide+5mm-tris buffer to 0.25m-sucrose for use as a suspending medium maintained the sum of the NADP+NADPH(2) concentrations in homogenates at the level found in intact tissue for at least 30min. at 0 degrees . 2. The effects of freezing intact tissue and homogenates in liquid nitrogen before the extraction of NADP and NADPH(2) were studied. Freezing alone appears to convert a significant amount (approx. 30%) of liver NADPH(2) into an equivalent amount of NADP in intact tissue. This is discussed in terms of the ;bound NADP' reported by Burch, Lowry & Von Dippe (1963). 3. The intracellular distributions of NADP and NADPH(2) in intracellular fractions of rat liver were studied by using a modified centrifuging scheme that allows extraction of the isolated fractions to be performed within 45min. of killing the animal. Approx. 50% of the total NADP+NADPH(2) was found in the large-particle fractions and the remaining 50% was mostly in the soluble fraction of the cell. 4. Further investigations are reported on the nature of ;bound NADP' in rat liver. Most of this material appears associated with the ;nuclear' (containing nuclei, debris, erythrocytes etc.) or large-mitochondrial fractions, or both, obtained by low-speed centrifuging of rat-liver homogenates. 5. Although in some experiments the variations produced in the concentration of NADPH(2) present in large-particle fractions were followed by similar changes in that of ;bound NADP', in other cases no such direct relationship was obtained. Addition of phenazine methosulphate, for example, consistently lowered the concentration of NADPH(2) yet raised the concentration of ;bound NADP' in rat-liver mitochondrial fractions.