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High pH ammonia toxicity, and the search for life on the Jovian planets.

Jovian plants have enviroments apparently suitable for the evolution of life, but nevertheless, present severe challenges to organisms. One such challenge arises from the presence of ammonia. Ammonia is an efficient biocide, its effect being dependent on pH as well as on concentration. The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural enviornments of high pH and/or ammonia concentration. Escherichia coli and Bacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, survival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is, nevertheless, two to three orders of magnitude longer than for E. coli. Our data support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.

Ammonia

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method.

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.

Alcohols

Coil planet centrifugation and its application to the observation of altered membrane properties of erythrocytes in hepatobiliary disorders.

A coil planet centrifuge (CPC) is an apparatus aiming to obtain a centrifugal force exerting on particles suspended in a fluid in a coiled polyethylene tube (coil) which makes self-rotation around its axis and revolution around the principal axis simultaneously. If a linear osmotic gradient is prepared in the coil and a small amount of blood is applied to it and subjected to centrifugation, one is able to observe the membrane properties of erythrocytes in terms of the shift of hemolysis band appearing in the coil with high accuracy and precision. Coil planet centrifugation was applied to the observation of altered membrane properties of erythrocytes in hepatobiliary diseases. Remarkable shifts of end-point of the hemolysis band going in parallel with the severity of damage were visualized. Study as to its cause revealed that increased free cholesterol content of erythrocyte membrane which was the result of plasma cholesterol Esterstürz due to inhibition of lecithin-cholesterol acyltransferase by stagnant bile acids was responsible for this phenomenon. Elevation of serum bile acid concentration and increased serum leucine-aminopeptidase were closely correlated to the shift of hemolysis end-point toward the low osmolal side. CPC may be useful for detection and chasing the clinical course of patients with hepatobiliary disorders.

Acute Disease

Hemolytic pattern of erythrocytes in the newborn measured by the coil planet centrifuge system and its relationship to neonatal jaundice.

Dynamic osmotic fragility of erythrocytes in cord blood and its changes during the neonatal period were studied by means of a coil planet centrifuge system. The starting-point for hemolysis in the newborn become similar to that of adult blood after approximately a week, while the shift of the end-point to the adult range required one month or more. The percentage of fragile cord blood cells with a hemolysis starting-point above 110 m osmol and the maximum bilirubin level during neonatal period were examined. A high percentage of fragile cells was associated with high bilirubin levels, and when fragile cells comprised more than 7.0% of the total cord blood erythrocytes, the bilirubin level tended to rise above 15 mg/dl.

Bilirubin

A proposal concerning the origin of life on the planet earth.

The widely accepted Oparin thesis for the origin and early evolution of life seems sufficiently far from the true state of affairs as to be considered incorrect. It is proposed that life on earth actually arose in the planet's atmosphere, however an atmosphere very different from the present one. Because of an extremely warm surface, the early earth may have possessed no liquid surface water, its water being partitioned between a motten crust and a fairly dense atmosphere. Early preliving systems are taken to arise in the droplet phase in such an atmosphere. The early earth, which resembled Venus then and to some extent now, underwent a transition to its present condition largely as a result of the evolution of methanogenic metabolism.

Animals

Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge.

Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.

Centrifugation

Preliminary applications of cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography.

The cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius and a total capacity of 1600 ml was successfully applied to preparative counter-current chromatography of various biological samples, which include sea buckthorn extract, steroid reaction mixture, indole plant hormones, and dinitrophenylamino acids. The present system offers advantages of stable balance of the centrifuge, a large column capacity, and high resolution.

Amino Acids

Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography. I. Apparatus and studies on stationary phase retention in short coils.

Using a new cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius, the retention of the stationary phase for nine solvent systems was studied with short coils mounted at two different locations on three holders with 5-, 15- and 25-cm hub diameters. Coils mounted 10 cm to the left of the center of a holder produced a much improved retention of most of the solvent systems compared with the same coils mounted at the center of the holder. In the lateral coil position the retention was found to be affected by the direction of the planetary motion and the head-tail elution mode. This phenomenon may be attributed to the effect of the lateral force field acting asymmetrically between the upper and lower halves of the coil.

Centrifugation

Foam counter-current chromatography with the cross-axis synchronous flow-through coil planet centrifuge.

A novel coil planet centrifuge system performs efficient foam separation in a long multilayer coil. Potential capability of the method was successfully demonstrated in separations of two sets of test samples using sodium dodecyl sulfate as a foaming agent. Rhodamine B with high foam affinity was quickly separated from Evans blue lacking foam affinity. A high concentration of salts such as disodium hydrogenphosphate in the surfactant solution effectively lowered the foam affinity of basic dyes, yielding complete peak resolution of rhodamine B and methylene blue according to a subtle difference in foam affinity.

Centrifugation

Cross-axis synchronous flow-through coil planet centrifuge for large-scale preparative counter-current chromatography. III. Performance of large-bore coils in slow planetary motion.

A preparative capability of the present cross-axis synchronous flow-through coil planet centrifuge was demonstrated with 0.5 cm I.D. multilayer coils. Results of the model studies with short coils indicated that the optimal separations are obtained at low revolutional speeds of 100-200 rpm in both central and lateral coil positions. Preparative separations were successfully performed on 2.5-10 g quantities of test samples in a pair of multilayer coils connected in series with a total capacity of 2.5 l. The sample loading capacity will be scaled up in several folds by increasing the column width.

Amino Acids

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography. I. Design of the apparatus and analysis of acceleration.

A novel design of the cross-axis synchronous flow-through coil planet centrifuge is introduced. The apparatus holds a pair of large coil holders symmetrically, one on each side of the rotary frame, at a lateral position 12.5 cm from the center of the holder shaft held 10 cm from the centrifuge axis. Mathematical analysis of acceleration generated by the planetary motion of the apparatus revealed a unique centrifugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.

Chromatography

New angle rotor coil planet centrifuge for counter-current chromatography. I. Analysis of acceleration.

Simple mathematical analysis has been performed on a new type of synchronous planetary motion to elucidate the acceleration field. Using generalized formulae derived from the above analysis, distribution of the centrifugal force vectors produced by all types of the synchronous planetary motion is computed and expressed in the same format to facilitate comparative studies. The results of the above analysis predict high performance of the new angle rotor coil planet centrifuge in counter-current chromatography.

Centrifugation

New angle rotor coil planet centrifuge for counter-current chromatography. II. Design of the apparatus and studies on phase retention and partition capability.

A new angle rotor coil planet centrifuge (psi = 25 degrees), which produces a Type J-L synchronous planetary motion, has been constructed to examine its capability in terms of stationary phase retention and solute partitioning. Studies on phase distribution diagrams obtained from various two-phase solvent systems indicated that the present system can be adapted to a wide variety of solvent systems by adjusting the centrifugal conditions. Excellent partition capability of the apparatus was successfully demonstrated in separations of dinitrophenyl amino acid samples with chloroform-acetic acid-0.1 N hydrochloric acid (2:2:1).

Amino Acids

Cross-axis synchronous flow-through coil planet centrifuge (type XLL). II. Speculation on the hydrodynamic mechanism in stationary phase retention.

The hydrodynamic mechanism involved in the retention of stationary phase in the present x-axis coil planet centrifuge system is discussed. A statistical treatment of the retention data disclosed important clues such as the effect of the inward-outward elution mode, the close correlation between the planetary motion and the head-tail elution mode, and the superior retention capacity of the left-handed coils. The combined effects of the radial and lateral centrifugal force field derived from the mathematical analysis of acceleration acting on the coil provide an explanation for the phenomena.

Centrifugation

Semi-preparative purification of an endogenous ligand for brain serotonin-2 receptors by coil planet centrifuge counter-current chromatography.

A horizontal flow-through coil planet centrifuge equipped with a rotatory frame holding three sets of composite column assemblies was used for purification of an endogenous ligand (ketanserin binding inhibitor) for the [3H]-ketanserin (3H-KET) recognition site. The protein mixture containing the endogenous material was successfully resolved by using a two-phase solvent system consisting of 95% ethanol-31.5% ammonium sulphate (1:2). The active fractions on 3H-KET binding obtained after counter-current chromatography (CCC) were further purified through a C18 microBondapak reversed-phase high-pressure liquid chromatographic column. The introduction of this advanced CCC technique represents an important step in the application of CCC for the separation of polar proteins from protein mixtures.

Ammonium Sulfate

Studies on the preparative capability of the horizontal flow-through coil planet centrifuge and high-performance liquid chromatography in the separation of polar compounds from Oxytropis ochrocephala Bunge.

Horizontal flow-through coil planet centrifuge (CPC) and high-performance liquid chromatography (HPLC) techniques were used for separation of polar compounds from a crude ethanol extract of Oxytropis ochrocephala Bunge, a poisonous legume plant widely distributed in northwestern China. The performance of these two chromatographic methods was compared in terms of column efficiency, peak resolution, separation time, sample loading capacity, etc. The results indicated that two polar compounds in the crude extract were equally well separated by these two methods. HPLC gave comparable peak resolution in shorter separation time while its sample loading capacity was limited to the mg range. The CPC method required a long separation time, but yielded a higher purity of fractions with a much greater capacity.

Centrifugation

Osmotic fragility changes in preserved blood: measurements by coil planet centrifuge and parpart methods.

The coil planet centrifuge (CPC) can be used to measure the osmotic fragility of erythrocytes. Fragility measured by this method alters when different salts are used. The CPC and Parpart methods were used to measure the changes during storage in red cell osmotic fragility in ACD or CPD blood with or without adenine. More marked changes were detected by the CPC method, especially in old cells. The changes of fragility of erythrocytes during storage seem to occur mainly in old cells. Adenine is effective in preventing such changes.

Adenine