Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients,an improved,simplified method

A mathematical method is described for calculating the sedimentation coefficient (s_{20, w}) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evalution (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.     

Defective Bacteriophage PBSH in Bacillus subtilis: I. Induction, Purification, and Physical Properties of the Bacteriophage and Its Deoxyribonucleic Acid

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.     

Sedimentation velocity, multi-speed method for analyzing polydisperse solutions

A method is described for the sedimentation velocity analysis of solutions composed of macromolecular solutes of widely disparate size. In sedimentation velocity experiments, usually a single rotor speed is chosen for the entire run, and consequently, the range of observable sedimentation coefficients can be severely limited. This limitation can be removed if the speed is varied during the run, starting with a relatively low speed so that the largest particles can be easily observed. The speed is increased during the run until full speed is attained and the run continued at full speed until the smallest species of interest have cleared the solution. The method, called wide distribution analysis, is based on the method developed originally by Yphantis and co-workers (Proc. Natl. Acad. Sci. USA 78 (1981) 1431) and on the time derivative method of Stafford (Anal. Biochem. 203 (1992) 295), essentially eliminating both the time-independent and radially-independent noise thereby improving the precision, especially for interference optics. An algorithm for analysis of data from both absorbance and interference optics and experimental protocols compatible with the Beckman XL-I Analytical Ultracentrifuge are presented. With these protocols an extremely wide range of sedimentation coefficients from approximately 1.0 to 250000 S can be accommodated in a single multi-speed run.     

ECHINOID AND CRUSTACEAN BURROWS AND THEIR DIAGENETIC SIGNIFICANCE IN THE MAASTRICHTIAN-DANIAN OF STEVNS KLINT, DENMARK

The increase in species and specimens of fossils in the uppermost part of the Maastrichtian White Chalk is interpreted as a result of reduced depth. The absence of bryozoans, brachiopods, and regular echinoids in the Cerithium Limestone indicates sedimentation in tidal pools. After sedimentation of the Cerithium Limestone, burrowing activity followed. A burrow of Brissopneustes danicus similar to burrows of the recent Echinocardium cordatum is described. Callianassa and its burrows are found in the Upper Danian calcarenite but not in the Lower Danian or Maastrichtian of Denmark. The dominant type of burrows along the Maastrichtian-Danian boundary has presumably been formed by the crustacean Ctenocheles.
The early post-Maastrichtian burrowing activity was succeeded by (1) induration of the bottom sediment and a slight abrasion (2) dissolution of aragonite shells and siliceous sponges, (3) offshore sedimentation and filling of the burrows with Lower Danian chalk mud, bryozoan fragments and other fossil remains, and (4) settling in the deeper part of the soft chalk sediment and precipitation of flint in or around burrows near the surface of the sediment.     

Plant community and landscape patterns of a floodplain wetland in Maputaland, Northern KwaZulu-Natal, South Africa

The lower Mkuze River floodplain is located east of the Lebombo Mountains on the Maputaland Coastal Plain in northern KwaZulu‐Natal, South Africa. The vegetation ecology of the floodplain was examined using the hierarchical framework described by landscape ecology theory. The smallest spatial scale to which the vegetation of the floodplain was described was the relatively homogeneous units of plant communities. From a landscape ecology perspective this level of analysis is referred to as the grain. Six plant communities were identified using two‐way indicator species analysis (TWINSPAN) classification. The distribution of these plant communities were correlated to an underlying inundation‐sedimentation gradient using the ordination technique, detrended correspondence analysis (DCA). This correlation provided a useful foundation for the examination of ecological processes and phenomena at the next higher, spatially coarser level within the landscape hierarchy, namely the reference level. This reference level was described by three functional types delimited by differing flooding and sedimentation regimes. The use of landscape ecology theory guided the interpretation of results by explicitly recognizing the importance of spatial heterogeneity, hierarchical organization and dynamics, and proved invaluable in developing process‐based understanding of the vegetation ecology of the lower Mkuze River floodplain.     

Band centrifugation of macromolecules in self-generating density gradients. II. Sedimentation and diffusion of macromolecules in bands

Three approaches to the simultaneous sedimentation and diffusion of hands or zones of noninteracting homogeneous macromolecules are examined: (1) The authors' method of moments: (2) the transport me of Sehumaker and Rosenbloom; and (3) the stochastic solution of the Lamm equation due to Gehatia and Katehalski. All three methods indicate that the motion of the maximum of the hand may be used to evaluate the sedimentation coefficient. The moment, method provides relations which appear to be useful for measuring diffusion coefficients. Relations are given for the analysis of resolved components. The problem of measuring sedimentation coefficients of macromolecules with concentration-dependent sedimentation coefficients is examined. Methods are described for evaluating the sedimentation coefficient in these systems and for obtaining the sedimentation coefficient at infinite dilution. Methods are described for determining the weight-average sedimentation coefficient in Multi-component systems, and the differential and integral distribution of sedimentation coefficients of macromolecules with low-diffusion coefficients.     

Separation of megakaryocytes from rat bone marrow cells using velocity sedimentation in an isokinetic gradient of ficoll in tissue culture medium

A technique for the purification of rat megakaryocytes is described. Velocity sedimentation in a previously described isokinetic gradient of Ficoll (polysucrose) in tissue culture medium was more effective than isopycnic sedimentation for the purification of megakaryocytes and resulted in preparations of megakaryocytes which contained 2.4 ± 0.8% (range 1.85–3.60%) megakaryocytes. Megakaryocytes exhibited a broad range of density between 1.06 and 1.15 gm/ml. The inaccuracy which is inherent in the use of velocity sedimentation without isopycnic sedimentation as a means of particle size analysis is discussed.     

Asymmetrical soft-sediment deformation structures triggered by rapid sedimentation in turbiditic deposits (Late Miocene, Guadix Basin, southern Spain)

Summary Soft-sediment deformation structures in Tortonian turbiditic deposits of the Guadix Basin (southern Spain) have been described. The most common structures are asymmetrical pillow structures and elongated sets of loadcasts. The structures are metric in scale and have been interpreted as the result of liquefaction and/or fluidization processes triggered by the rapid sedimentation of single high concentration turbidites. Final morphology of soft-sediment deformation structures is related to two main driving force systems: unstable density gradient and lateral shear stress. The latter is probably induced by the downslope component of the sediment weight. The asymmetry of deformational structures (in horizontal and vertical cross-section) allows a clarification of the relationship between morphology of deformation and direction of lateral shear stress: this relationship seems ambiguous and confused in the literature. The interpretations both of deformation mechanism and trigger agent have been supported with:-field analyses;-calculations on the liquefaction processes induced by rapid sedimentation;-qualitative models in laboratory.     

Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.     

Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.     

Nucleosome mono, di, tri-, and tetramers from chicken embryo chromatin.

The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0-1 M KC1 that decreases with increasing repeat number. Less histone H1 is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same Tm of 78 degrees C has been determined for the KC1-soluble nucleosomes and for the KC1-insoluble monomer. The Tm of the KC1-insoluble oligomers is 79.8 degrees C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4 degrees C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KC1) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the sedimentation and molecular weight data of KC1-soluble nucleosome mono- and tetramers.     

Some impacts of human activities on trout, Salmo trutta, populations

SUMMARY. 1. The life cycle of salmonid fishes is described.
2. The performance and environmental requirements of the various life stages of the trout ( Salmo trutta L.) are reviewed, (a) The literature gives predictive relationships between water temperature and rate of embryonic development, food requirements and growth rate, (b) Water temperature, intragravel oxygen supply rate, water pH, the occurrence of mechanical shock, disturbance of spawning gravels, sedimentation and water chemistry can all influence the survival of the intragravel stages, (c) The survival and/or well-being of the free-swimming stages and the success of spawning are influenced by such factors as dissolved oxygen concentration, pH, water depth, water velocity and water chemistry.
3. Human activities such as impoundment, river transfer, drainage works, land improvement, afforestation and deforestation can all influence trout populations via changes in flow regime (and related effects such as sedimentation), temperature regime and water chemistry.
4. Man can also influence trout populations directly by cropping for food and/or sport and by artificial stocking.
5. Examples of practical application of present knowledge are given and some future research needs are listed.     

Distinguishing cytomegalovirus, mycoplasma, and cellular DNA polymerases.

Cytomegalovirus-induced DNA polymerase can be distinguished from infected-cell enzymes by activity in 100 mM (NH4)2SO4. Virus polymerase is stimulated to 145% of control, whereas mock-infected cell polymerase is inhibited to 12% of control without added salt. Mycoplasmas induce a DNA polymerase in cell extracts that is stimulated to 130 to 180% by 25 mM (NH4)2SO4. Mycoplasma DNA polymerase may be mistaken for a virus-induced polymerase when virus stocks are contaminated. Identification of virus, cellular, and mycoplasma DNA polymerases in total cell extracts is described using sedimentation rate and effect of inhibitors on DNA polymerase activities.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Sedimentation velocity of sodium hyaluronate-lysozyme mixtures

A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s_20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold.     

Self-association of rabbit muscle phosphofructokinase at pH 7.0: stoichiometry

The self-association of rabbit muscle phosphofructokinase at pH 7.0 was investigated by velocity sedimentation. The process was demonstrated to be in a rapid, dynamic equilibrium. The concentration dependence of the weight-average sedimentation coefficient was monitored within the range of 10-750 microgram/mL. The sedimentation properties of phosphofructokinase were analyzed by theoretical simulations or an associating system in rapid equilibrium. In the absence of any ligands and at a temperature of 23 degrees C, the simplest computed model which gives the best fit between theoretical and experimental points can be described as progressive association of monomer in equilibrium or formed from tetramer in equilibrium or formed from 16-mer with apparent equilibrium constants K4 = 5.06 X 10(5) (mL/mg)3 and K16 = 3.25 X 10(23) (mL/mg)15. However, at 5 degrees C, the equilibrium was altered and can best be described as monomer in equilibrium or formed from dimer in equilibrium or formed from tetramer in equilibrium or formed from 16-mer.     

Nucleic acid chemistry: resolution of supercoiled, intact relaxed, and nicked double-stranded DNAs by formamide-sucrose gradients

A technique which will separate superhelical, intact nonsuperhelical, and damaged mitochondrial DNA molecules by use of sedimentation gradients of formamide-sucrose is described. This technique can be used to detect damage to the mitochondrial DNA helix.     

The role of <Emphasis Type="Italic">Viviparus contectus</Emphasis> (Millet) (Mollusca,Viviparidae) in the sedimentation of suspension and transformation of organic matter in the Tnya River (Ukraine)

As active filterers and sedimentators, mollusks Viviparus contectus (Millet) clear water from suspensions in the Tnya River. The sedimentation ability of mollusks is assessed on the basis of experimental studies at three stations using the funneling technique. Mollusks at the age of 3 and 5 years are used. The rate of sedimentation of V. contectus is 7.3 to 13.7 mg/(spec. day) in July 2014. The efficiency of suspended sedimentation is higher in younger individuals. The rate of sedimentation is determined by the total body weight of mollusks; the ratio between these parameters is described by the exponential function. V. contectus contributes to the decrease in the content of organic matter in the water with a rate of 0.055 to 0.069 mg of O₂/(dm³ spec. h), as well as to the enrichment of the near-bottom water layer with organic matter.     

Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.