A comparison of two centrifuge techniques for constructing vulnerability curves: insight into the ‘open‐vessel’ artifact

A vulnerability curve (VC) describes the extent of xylem cavitation resistance. Centrifuges have been used to generate VCs for decades via static‐ and flow‐centrifuge methods. Recently, the validity of the centrifuge techniques has been questioned. Researchers have hypothesized that the centrifuge techniques might yield unreliable VCs due to the open‐vessel artifact. However, other researchers reject this hypothesis. The focus of the dispute is centered on whether exponential VCs are more reliable when the static‐centrifuge method is used rather than the flow‐centrifuge method. To further test the reliability of the centrifuge technique, two centrifuges were manufactured to simulate the static‐ and flow‐centrifuge methods. VCs of three species with open vessels of known lengths were constructed using the two centrifuges. The results showed that both centrifuge techniques produced invalid VCs for Robinia because the water flow through stems under mild tension in centrifuges led to an increasing loss of water conductivity. In addition, the injection of water in the flow‐centrifuge exacerbated the loss of water conductivity. However, both centrifuge techniques yielded reliable VCs for Prunus, regardless of the presence of open vessels in the tested samples. We conclude that centrifuge techniques can be used in species with open vessels only when the centrifuge produces a VC that matches the bench‐dehydration VC.     

Sedimentation coefficient of T-even bacteriophage DNA

The sedimentation coefficient of T2 phage DNA has been studied by zone centrifugation in sector-shaped preparative centrifuge tubes over a concentration range of 0.02–2.0 μg./ml. DNA. These results have been compared to a similar study in the analytical centrifuge of T4 DNA over the range of 0.50–5.75 μg./ml. DNA. A value for the sedimentation coefficient of 60.7 ± 1.8 S. was obtained by the first method and a value of 61.3 ± 1.5 S. by the second.     

A new type of cytocentrifuge,the valve-centrifuge

Summary A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves.The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation.Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei through gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.     

ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry.     

Computer simulations of mass transport for nonidentical interacting molecules.

We have developed a system for simulating the mass transport properties of interacting nonidentical macromolecules where the association is of the form A + B ? C, C + B ? D. Our simulation programs operate in a minicomputer (PDP 1104) with 16K of core and provide results identical to methods previously usable only with large computers. We use a rectangular approximation for the centrifuge cell which greatly simplifies calculation, although it introduces a few percent error into any attempt at quantitative fitting of actual data. The program as written is directly applicable to gel chromatography, simply by substitution of flow for centrifugal field and dispersion for diffusion. Simulations of centrifuge results have been compared with experimental results for two systems which have been proposed to fit the association pattern described—nitrogenase components and an antigen-antibody interaction. In both cases the results of our simulations suggest that the accepted interpretation of the experimental results may need to be modified. For the antigen-antibody interaction, the presence of multivalent higher order complexes apparently is required to explain the centrifuge results. For nitrogenase, one cannot readily distinguish the case of association to form both 1:1 and 1:2 molar complexes from that of formation of only the 1:1 complex on the basis of the published data. Criteria for making such a discrimination are discussed.     

Vulnerability curves by centrifugation: is there an open vessel artefact,and are ‘r’ shaped curves necessarily invalid?

Vulnerability curves using the 'Cavitron' centrifuge rotor yield anomalous results when vessels extend from the end of the stem segment to the centre ('open-to-centre' vessels). Curves showing a decline in conductivity at modest xylem pressures ('r' shaped) have been attributed to this artefact. We determined whether the original centrifugal method with its different rotor is influenced by open-to-centre vessels. Increasing the proportion of open-to-centre vessels by shortening stems had no substantial effect in four species. Nor was there more embolism at the segment end versus centre as seen in the Cavitron. The dehydration method yielded an 'r' shaped curve in Quercus gambelii that was similar to centrifuged stems with 86% open-to-centre vessels. Both 'r' and 's' (sigmoidal) curves from Cercocarpus intricatus were consistent with each other, differing only in whether native embolism had been removed. An 'r' shaped centrifuge curve in Olea europaea was indistinguishable from the loss of conductivity caused by forcing air directly across vessel end-walls. We conclude that centrifuge curves on long-vesselled material are not always prone to the open vessel artefact when the original rotor design is used, and 'r' shaped curves are not necessarily artefacts. Nevertheless, confirming curves with native embolism and dehydration data is recommended.     

Sedimentation Rate as a Measure of Molecular Weight of DNA

Zone centrifugation of mixtures of two labeled DNA's at low concentrations in density gradients of sucrose permits accurate measurement of relative sedimentation rates. The individual rates are constant during the run. Measurements with DNA's from phages T2, T5, and lambda conform to the relation D₂/D₁ = (M₂/M₁)^0.35, where D and M refer to distances sedimented and molecular weights of the DNA pair. The results show that high molecular weight DNA's sediment artificially fast in the optical centrifuge, owing to a hitherto unknown effect of molecular interactions. The molecular weight of lambda DNA is 31 million, measured either from sedimentation rate or from tests of fragility under shear.     

Morphology of Arabidopsis Grown under Chronic Centrifugation and on the Clinostat

Morphological measurements were made on populations of Arabidopsis thaliana grown from seed for 21 days under essentially constant environmental conditions except for the influence of gravitational or centrifugal accelerations. Growth conditions were what had been proposed for experiments in an artificial satellite. Observations are reported for plants grown at normal 1-g upright or on horizontal clinostats and for plants grown on a centrifuge. Increased g-force, up to 15 times normal, was found to have significant but small effects on some morphological end points. The plants' sensitivity to the magnitude of the g-force was much less than to its vector direction.     

Intermediates of replication of NR1 plasmid DNA in Escherichia coli mini-cells

The pulse label of NRL plasmid-containing mini-cells has been shown to be localized mainly in DNA with a floating density in the CsCl-EtBr gradient different from the floating density of supercoil and open circle DNAs. During the chase of the pulse label, the DNA is transfered from the fraction with the intermediate floating density varying between the values for the supercoil and open circle DNA fractions to the fraction located below supercoil DNA in the equilibrium gradient and further to the open circle fraction. Electron microscopic analysis of the material with a higher floating density as compared to supercoil DNA has demonstrated the presence of "heavy" intermediates--covalently closed loosely supercoiled molecules. It is also supported by the sedimentation pattern of the characterized fraction in neutral and alkaline saccharose gradients. Molecules located in the CsCl-EtBr gradient between supercoil and open circle DNAs have the sedimentation constant characteristic for the elongation intermediates. It is suggested that NRL DNA molecules in E. coli mini-cells pass through all the basic stages of replication which results in the formation of open circle DNA or supercoil relaxation complexes.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

Theory of sedimentation for ligand-mediated heterogeneous association-dissociation reactions

Theoretical analytical sedimentation patterns have been computed for ligand-mediated heterogeneous association-dissociation reactions between macromolecules. Involvement of either a single kind of ligand or two different ligands acting in a stepwise fashion has been considered. Self-association, mediated in a stepwise fashion by two different ligands, has also been examined. The conclusion reached is that such interactions have the potentiality for exhibiting as many as three or four sedimenting peaks despite rapid rates of reaction. In general, the peaks correspond to different equilibrium compositions and not to individual macromolecular species; that is to say, they constitute a reaction boundary. Their resolution depends upon generation of concentration gradients of ligand(s) along the centrifuge cell by chemical reequilibration during sedimentation of the several macromolecular species. The implications of these findings for fundamental studies on subunit proteins and protein assemblies and for conventional applications of ultracentrifugation are discussed.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

15. Investigations of Poliomyelitis Antigens

ABSTRACT

The complex nature of the antigens in suckling mouse-adapted MEFi virus was indicated by Selzer and Poison¹, who found that the infectivity was associated with two components of approximate sedimentation coefficients 100 and 173 S respectively. In addition to these components a “soluble antigen” was found which had a sedimentation coefficient of about 22 S. These results, which were obtained by using a Spinco preparative centrifuge (Poison and Linder²), have now been confirmed in the Spinco analytical ultracentrifuge of the Nobel Institute, Stockholm, and other types of poliomyelitis virus have been studied in both instruments.     

Chasing the Patagonian sun: comparative thermal biology of Liolaemus lizards

The importance of the thermal environment for ectotherms and its relationship with thermal physiology and ecology is widely recognized. Several models have been proposed to explain the evolution of the thermal biology of ectotherms, but experimental studies have provided mixed support. Lizards from the Liolaemus goetschi group can be found along a wide latitudinal range across Argentina. The group is monophyletic and widely distributed, and therefore provides excellent opportunities to study the evolution of thermal biology. We studied thermal variables of 13 species of the L. goetschi group, in order to answer three questions. First, are aspects of the thermal biology of the L. goetschi group modelled by the environment or are they evolutionarily conservative? Second, have thermal characteristics of these animals co-evolved? And third, how do the patterns of co-evolution observed within the L. goetschi group compare to those in a taxonomically wider selection of species of Liolaemus? We collected data on 13 focal species and used species information of Liolaemus lizards available in the literature and additional data obtained by the authors. We tackled these questions using both conventional and phylogenetically based analyses. Our results show that lizards from the L. goetschi group and the genus Liolaemus in general vary in critical thermal minimum in relation to mean air temperature, and particularly the L. goetschi group shows that air temperature is associated with critical thermal range, as well as with body temperature. Although the effect of phylogeny cannot be ignored, our results indicate that these thermal biology aspects are modelled by cold environments of Patagonia, while other aspects (preferred body temperature and critical thermal maximum) are more conservative. We found evidence of co-evolutionary patterns between critical thermal minimum and preferred body temperature at both phylogenetic scales (the L. goetschi group and the extended sample of 68 Liolaemus species).     

Sediment and Turbidity Associated with Offshore Dredging Increase Coral Disease Prevalence on Nearby Reefs

In recent decades, coral reef ecosystems have declined to the extent that reefs are now threatened globally. While many water quality parameters have been proposed to contribute to reef declines, little evidence exists conclusively linking specific water quality parameters with increased disease prevalence in situ. Here we report evidence from in situ coral health surveys confirming that chronic exposure to dredging-associated sediment plumes significantly increase the prevalence of white syndromes, a devastating group of globally important coral diseases. Coral health surveys were conducted along a dredging-associated sediment plume gradient to assess the relationship between sedimentation, turbidity and coral health. Reefs exposed to the highest number of days under the sediment plume (296 to 347 days) had two-fold higher levels of disease, largely driven by a 2.5-fold increase in white syndromes, and a six-fold increase in other signs of compromised coral health relative to reefs with little or no plume exposure (0 to 9 days). Multivariate modeling and ordination incorporating sediment exposure level, coral community composition and cover, predation and multiple thermal stress indices provided further confirmation that sediment plume exposure level was the main driver of elevated disease and other compromised coral health indicators. This study provides the first evidence linking dredging-associated sedimentation and turbidity with elevated coral disease prevalence in situ. Our results may help to explain observed increases in global coral disease prevalence in recent decades and suggest that minimizing sedimentation and turbidity associated with coastal development will provide an important management tool for controlling coral disease epizootics.     

Use of operative temperature and standard operative temperature models in thermal biology

Operative temperature (T_e) and standard operative temperature (T_es) models have been used to address ecological questions about the thermal biology of ectotherms and endotherms for over 25 years. This review focuses on the accuracy and use of T_e and T_es models in ecological and physiological studies. The utility of T_e and T_es models lie in the fact that they take a multivariate problem involving inputs of air temperature, ground temperature, solar radiation, and wind speed and map them into a single thermal metric on a spatial scale appropriate for the animal. The most reliable T_e models are copper casts that mimic the morphology and absorptivity of an animal. Simplified T_e models such as cylinders and spheres have been shown to produce errors in T_e as large as 12 °C under certain conditions and should only be used after careful calibration against a live animal. The accuracy of heated T_es models has been addressed in much less detail then that of T_e models. When calibrated and used under conditions of low solar radiation, heated taxidermic mounts and simplified T_es models produce errors in net heat production on the order of 5% or less. In order to provide reliable data, all types of models must be calibrated over an ecologically realistic range of environmental conditions experienced by the animal. This advice has been largely ignored in the literature, where 61% of the of studies examined failed to properly calibrate the models prior to use. Additionally, studies using these models tend to lack experimental rigor, using only one or two models to make measurements on 1 or 2 days of the active season. When used correctly, T_e and T_es models can be powerful tools for integrating the thermal environment experienced by an animal into a single metric that can address questions regarding the ecology, physiology, and behavior of endotherms and ectotherms. However, until investigators make the effort to use these models in a scientifically valid manner with proper calibration and experimental design their value to thermal biologists will be limited.     

Diffusion of the Reaction Boundary of Rapidly Interacting Macromolecules in Sedimentation Velocity

Sedimentation velocity analytical ultracentrifugation combines relatively high hydrodynamic resolution of macromolecular species with the ability to study macromolecular interactions, which has great potential for studying dynamically assembled multiprotein complexes. Complicated sedimentation boundary shapes appear in multicomponent mixtures when the timescale of the chemical reaction is short relative to the timescale of sedimentation. Although the Lamm partial differential equation rigorously predicts the evolution of concentration profiles for given reaction schemes and parameter sets, this approach is often not directly applicable to data analysis due to experimental and sample imperfections, and/or due to unknown reaction pathways. Recently, we have introduced the effective particle theory, which explains quantitatively and in a simple physical picture the sedimentation boundary patterns arising in the sedimentation of rapidly interacting systems. However, it does not address the diffusional spread of the reaction boundary from the cosedimentation of interacting macromolecules, which also has been of long-standing interest in the theory of sedimentation velocity analytical ultracentrifugation. Here, effective particle theory is exploited to approximate the concentration gradients during the sedimentation process, and to predict the overall, gradient-average diffusion coefficient of the reaction boundary. The analysis of the heterogeneity of the sedimentation and diffusion coefficients across the reaction boundary shows that both are relatively uniform. These results support the application of diffusion-deconvoluting sedimentation coefficient distributions c(s) to the analysis of rapidly interacting systems, and provide a framework for the quantitative interpretation of the diffusional broadening and the apparent molar mass values of the effective sedimenting particle in dynamically associating systems.     

Mathematical Simulation of Dictyostelium Pseudoplasmodia Movements

A mathematical model has been developed to simulate the movement of Dictyostelium discoideum slugs. The model allows simulation of random motility in the absence of external stimuli based on spatial and temporal changes in the microenvironment. In addition the effects of external stimuli such as laterally impinging light and thermal gradients have been simulated. The model allows us to study the effect of simultaneous application of different stimuli. A statistical analysis incorporated in the model allows us to evaluate quantitatively the directedness of movement and the angle of deviation from the stimulus direction as well as the angular distribution of track segments in 5° sectors. The results are discussed in comparison with the experimental data obtained in vivo as well as the ecological effects of multiple stimuli.     

Ultracentrifugal studies in capillary cells. I. Determination of sedimentation coefficients

A technique has been developed which is based on transport method equations and allows determination of sedimentation coefficients using less than 5 ng of a biological active principle. Glass capillaries of 0.30 mm inside diameter and 3.0 mm length are used as sedimentation cells. About 200 nl of pure or impure solutions with concentration as low as 0.05 mg/ml are ultracentrifuged in a swinging-bucket rotor with a conventional preparative ultracentrifuge. The capillary microcells are easily sectioned after centrifugation through a plane previously marked on the glass surface. The technique was successfully extended to the use of larger glass capillaries, up to 1.25 mm inside diameter, and plastic centrifuge tubes of 5.0 mm diameter and 0.40 ml capacity. The method has been experimentally verified with proteins and protein-polysaccharides of known sedimentation constants.     

Immobilization antigens of Tetrahymena pyriformis : I. Assay and extraction

A method using immunodiffusion has been established to assay the two mutually exclusive temperature dependent immobilization antigens, H and T, of Tetrahymena pyriformis. Specific antiserum was obtained by exploitation of allelic or temperature induced variations among inbred strains for absorption of antisera prepared against whole cells. The antigens were extracted both from isolated cilia and from whole cell bodies. Mild detergent extraction was found to be more efficient than mechanical disruption of the cells by freeze-thawing. The sedimentation behavior in sucrose density gradients of active H antigen was the same, whether freeze-thaw or detergent extracted; similarly, the sedimentation behavior of T was the same following the two extraction methods. Extraction with acetic acid, as reported by others, solubilized the same material as the detergent, but the acid denatured the antigen. An estimate of the molecular weight of the antigen of 29 000 for H and 23 000 for T was made.