A gradient titration apparatus for determining spectrophotometric binding isotherms.

The construction of an automatic gradient titration apparatus using a multichannel peristaltic pump and a recording spectrophotometer is described. The ability of the apparatus to faithfully generate continuous spectrophotometric binding isotherms was tested in experiments studying the interaction of DNA with neutral red. The method has been shown to require low volumes of reactants, and complete binding curves can be produced in less than 15 min. The apparatus was also used to perform automatically the method of continuous variations in experiments determining the binding stoichiometry of calmagite and magnesium ion.     

A simplified high-resolution gradient analysis system

A simplified high-performance, semiautomatic apparatus for the analysis and fractionation of density gradient preparations is described. In this system the gradient, mounted directly under a standard recording spectrophotometer, is displaced upwards through a vertical straight-path flow cell, then downward to a fraction collector.The resolution attainable with this apparatus is greater than that required for the high fidelity analysis of small volume centrifuged density gradients. The system is easily adaptable to cesium gradients, zonal rotors, and to chromatographic columns where its high-resolution, bubble-free performance makes practical the analysis of both small and large volumes. It is significantly simpler and less costly to construct than other apparatus of comparable performance.     

Electrophoretic separation of cells in a density gradient.

A bulk electrophoretic method is described for the separation of mammalian cells in Ficoll-sucrose density gradients. The fractionation of cells is performed in a simple commercially available apparatus (Buchler Poly-Prep) which should facilitate the application of the technique in various laboratories. Details of the experimental procedure are presented along with typical model separations of erythrocytes from various species and mouse lymphocytes.     

Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient

Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.     

A simple microtechnique for isoelectric focusing on a density gradient.

Isoelectric focusing in a density gradient can be performed on chromatographic columns of 10–15-ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 10-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 110-ml column.With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.     

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).     

A universal gradient apparatus for multiple organic solvents. II. Fractionation and elution of serum lipids.

Chromatography of serum lipids on a silicic acid column by gradient elution and with continuous detection is described. The gradient is formed in a new organic gradient device based on the principle of E. Peterson and H. A. Sober ((1959) Anal. Chem.31, 857), starting from six initial organic solvents. The detection is realized by flame ionization on a LCM2 liquid chromatography detector (Pye-Unicam). The technique is less solvent- and time-consuming and is of interest in preparative separation and analysis of lipophilic substances.     

A new dynamometric technique to measure and locate endocranial structures. Preliminary report

An apparatus for measuring and recording the resistance to penetration of endocranial tissue has been planned and built. The probe carrier, driven by a constant-speed electric motor, is fitted onto a stereotactic head frame which is used to guide the tool to the intended target. The displacement and resistance encountered when the tool penetrates intracranial structures are measured and recorded on an x-y recorder. Preliminary tests performed on calf brain specimens have documented that the apparatus can measure the different consistencies of normal cerebral tissue and suggest a new technique for morphological investigations based on the mechanical consistency of normal and pathological organic tissue. Moreover, the hypothesis of a plastic deformation in cerebral tissue has been confirmed, in that the same apparatus permits one to measure the displacement of tissues caused by the advancement of the surgical tool.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles.

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.     

THE FINE STRUCTURE AND SCALE FORMATION OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA GEN. ET. SP. NOV.(CHRYSOLEPIDOMONADACEAE FAM. NOV., CHRYSOPHYCEAE)1

Chrysolepidomonas gen. nov. is described for single-celled monads with two flagella, a single chloroplast, and distinctive canistrate and dendritic scales. The type species, Chrysolepidomonas dendrolepidota sp. nov., is described for the first time. The canistrate scales bear eight “bumps” on the top surface, and the dendriticscales have a tapered base with a quatrifid tip. These organic scales are formed in the Golgi apparatus and storred in a scale reservoir. The scale reservoir is bounded on two sides by the R₁ and R₂ in microtubular roots of the basal apparatus. The cyst (=stomatocyst, statospore) forms endogenously by means of a silica deposition vesicle. The outer cyst surface is smooth, and the pore region is unornamented. Two other organisms bearing canistrate and dendritic scales, previously assigned to the genus Sphaleromants, are transferred to the genus Chrysolepidomonas. They are C.angalica sp. nov. and C. marine(Pienaar) comb. nov. The distinguishing features of Chrysolepidomonas and Sphaleromantis are discussed. A new family, Chrysolepidomonadceae fam. noc., is described for flagellates covered with organic scales.     

Design and construction of a preparative-scale dynamic field gradient focusing apparatus

A linear model is used to show that dynamic field gradient focusing (DFGF) can be scaled to preparative capacity, approximately O (10 mgs). This paper explains how the preparative-scale DFGF apparatus was designed and fabricated. Scaled-down experiments and mathematical modeling guided material selection and design changes during construction to increase the probability that the prototype preparative-scale DFGF apparatus would perform as intended. The finished prototype successfully focused bovine hemoglobin from an initial concentration of 6.82 to 15 mg/mL and allowed for 86% recovery of injected protein.     

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.     

Two-dimensional electrophoresis of plant proteins with phastsystem using nonequilibrium pH gradient separation.

We have adapted a two-dimensional electrophoretic technique described by P. Z. O'Farrell et al. (Cell 12, 1133-1142, 1977) to Phastsystem, resolving both acidic and basic proteins by using nonequilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. Protein separation was optimized for the analysis of plant proteins. The use of the Phastsystem apparatus reduced times of preparation and separation, allowing the rapid screening of plant proteins on a large scale of isoelectric points. This technique was used for the immunodetection and characterization of two stress-induced proteins in irradiated tomato leaves.     

Protein blotting in uniform or gradient electric fields

Experimental data and computer mapping were used to analyze electric fields generated by a variety of electrode arrays in protein blotting apparatus. Asymmetric electrode arrays were found to generate nonuniform fields that effected uneven transfers of 125I-labeled albumin from gels to nitrocellulose membranes. Symmetric arrays with multiple (four), independent wire electrodes, supplied individually with electric current, generated the most uniform fields and effected the most even transfers of the test protein. With multiple independent electrodes, gradient electric fields can be generated in which differences in electrophoretic elution between large and small proteins can be eliminated. Transfer apparatus with either uniform or gradient electric fields are expected to improve qualitative results and make possible quantitation of protein blotting.     

An apparatus for density gradient forming and nonpuncturing fractionation

A technique is described which allows gradient formation, sample layering, and nonpuncturing gradient fractionation in one apparatus. The demonstrated device can be adapted to centrifuge tubes of different sizes and volumes and connected to almost any device which is used for gradient formation or analysis with flow cuvettes and/or fraction collectors. The operation technique can be standardized. The construction gives reproducible gradients with sharp starting bands of the samples and provides good resolution after centrifugation.     

Mobility measurements by photometric analysis of zone electrophoresis in a sucrose gradient column

A density gradient zone electrophoresis apparatus has been designed for measuring electrophoretic mobilities on small amounts (about 10 μg) of viruses or other ultraviolet-absorbing materials. Dialysis membranes separate the gradient column from electrode chambers and eliminate the need to maintain hydrostatic equilibrium between the buffer chambers. The virus zone was located by pumping the gradient column through an ultraviolet flow cell, a procedure which did not disturb the virus zone. The apparatus was tested by measuring the mobilities of tobacco mosaic and brome mosaic viruses in different buffers, in sucrose, glucose, and glycerol gradients, at 0° and 15°, and during ascending and descending electrophoresis.     

黄土丘陵区表层土壤有机碳沿降水梯度的分布

沿368～591 mm降水量梯度选取7个调查地点、共63个调查样点,在每个样点选择恢复年限相近的林地、草地和农地,调查表层（0～30 cm）土壤有机碳的分布特征,分析气候、土层深度和土地利用类型等因素对土壤有机碳分布的影响.结果表明： 在黄土丘陵区368～591 mm的降水量范围内,表层土壤有机碳含量表现为草地（8.70 g·kg^-1）>林地（7.88 g·kg^-1）>农地（7.73 g·kg^-1）,土壤有机碳密度表现为草地（20.28 kg·m^-2）>农地（19.34 kg·m^-2）>林地（17.14 kg·m^-2）.林地、草地、农地的土壤有机碳含量无显著差异,综合3种土地利用类型的数据分析表明,不同降雨梯度下土壤有机碳含量差异显著（P<0.001）,土壤有机碳含量（r=0.838,P<0.001）与年均降水量间存在显著线性正相关关系;由北向南（以最北端鄂尔多斯为起点）,土壤有机碳含量沿着368～591 mm的年均降水量梯度的递增速率为0.04 g·kg^-1·mm^-1,土壤有机碳密度的递增速率为0.08 kg·m^-2·mm^-1.年均降水量、土壤黏粒含量、林下枯落物蓄积量和农作物根系密度可较好地模拟表层土壤有机碳分布.     

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.     

Protein desalting by isoelectric focusing in a segmented immobilized pH gradient

We have recently described an apparatus for protein purification based on a segmented Immobiline gel, having one or more liquid interlayers in between. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow chamber, and focusing the impurities in an Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. We now demonstrate that the present apparatus can be efficiently used for protein desalting. Hemoglobin A samples, containing 50 mM NaCl or 50 mM ammonium acetate, could be efficiently desalted in 2 h of recycling, after which the total salt content had decreased to less than 0.005 mM (a salt decrement of more than 10,000 fold the initial input). However, with polyprotic buffers (sulphate, citrate, phosphate, oligoamines) the desalting process was much slower, typically of the order of 20 h, possibly due to interaction of these species with the surrounding Immobiline matrix. In this last case, outside pH control (e.g. with a pH-stat) is necessary during protein purification, as, due to the faster removal of the monovalent counterion, the solution in the recycling chamber can become rather acidic or alkaline. It is demonstrated that the 2 extremities of the Immobiline segments facing the sample recycling chamber act indeed as isoelectric membranes, having a good buffering capacity, preventing the protein macroion from leaving the chamber by continuously titrating it to its isoelectric point.     

城市化梯度上亚热带常绿阔叶林土壤有机碳及其组分特征

以珠江三角洲城区(广州)、近郊(鼎湖山)及远郊(怀集)梯度上的亚热带常绿阔叶林为对象,研究不同土层土壤总有机碳、惰性有机碳、活性有机碳(易氧化有机碳、微生物生物量碳和水溶性有机碳)沿城市化梯度的变化,分析城市化对森林土壤有机碳的影响.结果表明: 0～5 cm土层土壤总有机碳和惰性有机碳含量均在城市化梯度上差异不显著,在5～60 cm土层,土壤总有机碳含量在远郊森林显著高于近郊和城区森林,而惰性有机碳含量在近郊森林最高,城区与远郊森林差异不显著.易氧化有机碳含量在近郊森林显著低于远郊(0～60 cm)和城区(0～10 cm)森林,微生物生物量碳含量在城区森林显著低于近郊和远郊森林,而水溶性有机碳含量在近郊森林(0～10 cm)显著低于城区森林.在0～20 cm土层,城区、远郊森林土壤活性有机碳占总有机碳的比例显著高于近郊森林,而惰性有机碳占总有机碳的比例在远郊森林最低;近郊与城区森林惰性有机碳占总有机碳的比例仅在5～10 cm差异显著.城市化增加了土壤活性碳组分,降低稳定性碳组分,不利于总有机碳积累,远郊森林土壤对城市化的响应更敏感.