Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

An integrating atomic absorption spectrophotometer for ultramicroanalysis of metals

An integrating atomic absorption spectrophotometer for ultramicroanalysis of metals is described. The apparatus is intended mainly for determination of calcium and magnesium but can also be used for sodium, potassium, lithium, and copper, among other metals. Analyses of sample volumes as small as about 1 nl, containing calcium and magnesium in amounts as low as 10^?11 and 10^?12 moles, respectively, have been made. In analyses of samples containing 4·10^?12 moles of magnesium a coefficient of variation of ±5% was obtained.The principles of the apparatus are the same as for a conventional atomic absorption spectrophotometer apart from the fact that an electronic integrator with a rapid response has been used as the recording system. For atomization of the sample a hydrogen-air flame has been used in combination with a flame adapter. The sample is introduced into the flame by means of a small loop on a platinum-iridium filament.     

Apparatus for continuous, large-volume suspended cell cultivation

A 30-liter apparatus for growing cells continuously is described. Samples are taken automatically, thus obviating one of the main causes of contamination. In order to eliminate the continuous introduction of a gas stream, the pH was kept constant by means of the injection of NH₄OH or NaOH. Under these circumstances, and by means of the two methods of agitation described—rotary and “by Vibromixer”—a quantity of cells of approximately 4 × 10¹⁰ is harvested every 48 to 72 hr.     

Quantitative evaluation of two pathways for ethanolamine phosphoglyceride biosynthesis in rat brainin vivo

[³H]Ethanolamine and [³²P]orthophosphate were injected intraventricularly into adult female rats. At varying time intervals after the injection (1–10 min), the animals were killed by means of a microwave apparatus, and phosphorylethanolamine and ethanolamine phosphoglycerides were extracted from the brains and counted after separation. The kinetic constants for phosphorylethanolamine incorporation into ethanolamine lipids were calculated both from³H data and from³²P data. From our results, it seems that base exchange reactions for ethanolamine incorporation into ethanolamine lipids are a pathway active in brainin vivo.     

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca²⁺-sensitive dyes is used for measurement of spatial and temporal aspects of Ca²⁺ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca²⁺-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca²⁺-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca²⁺ response as % change in fluorescence is obtained.     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.     

Conductometric apparatus for determining photosynthesic rate

A conductometric apparatus for determining the photosynthetic rate suitable predominantly for field measurements is described. In contrast with the hitherto described and applied instruments 3 samples of air can be analyzed here simultaneously, the data are temperature-independent and the entire electronic part of the apparatus is transistorized. The absorbers (300×30×30mm.) and the leaf chambers (140×100×10 mm.) are made of plexiglass. The membrane pump is hand-operated by means of a crank. The conductivity of the absorption solution (0.02N-KOH+0.5% isoamylalcohol+saponin) is measured in a special measuring cell into which the solution is transferred from the individual absorbers. The error of the photosynthetic rate determination is about 10-20% of the value obtained for intensities of 10-20 mg. CO₂ dm.-²h.-¹.     

Pressure probe technique for measuring water relations of cells in higher plants

A new method is described for continuously measuring cell turgor pressure (P), hydraulic conductivity (L_p), and volumetric elastic modulus (ε) in higher plant cells, using a pressure probe. This technique permits volume changes, ΔV, and turgor pressure changes, ΔP, to be determined with an accuracy of 10⁻⁵ to 10⁻⁶ μl and 3 to 5·10⁻² bar, respectively.

The main principle of the new method is the same as the pressure probe developed by Zimmermann and Steudle in which pressure is transmitted to a pressure transducer by means of an oil-filled capillary introduced into the cell. In order to use the pressure probe for small tissue cells, the effective compressible volume of the apparatus has to be sufficiently small in comparison to the volume of the cell itself. This is achieved by accurately fixing the oil/cell sap boundary in the very tip of the microcapillary by means of an electronic feedback mechanism, so that the effective volume of the apparatus is reduced to about 2 to 10% of the cell volume. In this way also, errors arising from compressibility of the apparatus and temperature fluctuations can be excluded.

Measurements on tissues cells of Capsicum annuum fruits yield ε values of 2 to 25 bar. Furthermore, ε can be shown to be a function of both cell turgor pressure and cell volume; ε increases with increasing turgor pressure and is higher in larger cells.

     

Studies on stimulus-response coupling in human neutrophils: II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na⁺-free, high-K⁺ buffered solution. The results demonstrate that: (a) the variation of Na⁺ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na⁺-free, high-K⁺ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na⁺ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.     

A chemical quenching apparatus for studying rapid reactions.

A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na⁺ + K⁺)-stimulated ATPase.     

Diazomethane: improved analytical and distillation techniques for small quantities and some synthetic applications

An improved design of apparatus for the small-scale (about 5 μmol to about 50 mmol) preparation of diazomethane is described. The diazomethane is generated from commonly used precursors and distilled by aeration in a glass apparatus connected by Teflon tubing and without a condenser. A new simple and reasonably accurate procedure for assay of diazomethane is described. This depends on reaction with excess [¹⁴C]benzoic acid in toluene followed by quantitative removal of the excess acid by partitioning with pH 10 aqueous buffer and assaying the methyl [¹⁴C]benzoate in the toluene by liquid scintillation counting. Examples are given of the use of accurately known amounts of diazomethane and [¹⁴C] diazomethane for the preparation of methylated derivatives of [2-¹⁴C]barbital, 4′-hydroxy-[2-¹⁴C]phenobarbital, and mephobarbital. Small amount(s) of dimethyl-barbital (O-methyl) were separated and partly characterized by gas chromatography/mass spectroscopy and NMR.     

A computer-controlled multi-titration system to study transpiration, OH− efflux and nitrate uptake by intact lettuce plants (Lactuca sativa L.) under different environmental conditions

Abstract An apparatus is described in which water and nutrient uptake (NO^?₃) and pH were measured and controlled by a microcomputer. Measurements were made twice hourly using appropriate electrodes, both in the nutrient solutions in which the plants were grown and in standard reference solutions. The signals from the electrodes were amplified to a range of 0–10 V and were fed to an A/D converter activated by the computer. The needs for water, NO^?₃, H⁺ and OH^? were then calculated and the necessary adjustments were made to the solutions by automatic injection of the correct amounts to each container in turn. These amounts were records of water and nitrate uptake and pH shift and were saved on disc. The plants were arranged on a turntable which gave additional benefit by smoothing out variations in light intensity. The system allowed a different solution to be maintained for each plant if required.     

New Solid-State Fermentation Chamber for Bulk Production of Aerial Conidia of Fungal Biocontrol Agents on Rice

A novel solid-state fermentation apparatus, namely an upright multi-tray conidiation chamber, was developed to facilitate the production of aerial conidia of fungal biocontrol agents, such as Beauveria bassiana. The chamber with 25 bottom-meshed metal trays had a capacity of ≥50 kg rice with each tray holding ≥2 kg. In repeated trials, a mean yield of 2.4 (1.8–2.7) × 10¹² conidia kg⁻¹ rice was harvested from the 7-day cultures of B. bassiana in a fully loaded chamber. The new apparatus has a high potential for bulk production of fungal conidia.     

Monoascorbate free radical-dependent oxidation-reduction reactions of liver Golgi apparatus membranes

Golgi apparatus from rat liver contain an ascorbate free radical oxidoreducatse that oxidizes NADH at neutral pH with monodehydroascorbate as acceptor to generate a membrane potential. At pH 5.0, the reverse reaction occurs from NAD⁺. The electron spin resonance signal of the ascorbate-free radical and its disappearance upon the addition of NADH (pH 7) or NAD⁺ (pH 5.0) confirms monodehydroascorbate involvement. Location of monodehydroascorbate both external to and within Golgi apparatus compartments is suggested from energization provided by inward or outward flux of electrons across the Golgi apparatus membranes. The isolated membranes are sealed, oriented cytoplasmic side out and impermeable to NAD⁺ and ascorbate. NAD⁺ derived through the action of Golgi apparatus β-NADP phosphohydrolase is simultaneously reduced to NADH with monodehydroascorbate present. The response of the NADH- (NAD⁺-) ascorbate free radical oxidoreductase system to pH in Golgi apparatus provides a simple regulatory mechanism to control vesicle acidification.     

The application of a laboratory apparatus for the study of nutrient fluxes between sediment and water

Sediments of the river Elbe estuary have been studied to assess their impact on the total nitrogen budget of the estuary. A new laboratory incubation apparatus was used to provide a means of regulating important parameters such as temperature and oxygen concentrations. With this apparatus sediment cores from a typical shallow water area with high organic carbon content were incubated under varying oxygen concentrations in the overlying water. Measurements of ammonium, nitrite, nitrate and nitrous oxide in the water phase were carried out and the fluxes between sediment and water phase calculated. During aerobic conditions in the water phase overall nitrate fluxes between + 4 and –3.5 mmol Nm^–2d^–1 across the sediment/water interface were observed. Under anaerobic conditions the fluxes increased up to –10 mmol Nm^–2 d^–1. Nitrous oxide was formed within the sediment under both aerobic and anaerobic conditions. Fluxes into the water phase were highest when the oxygen concentrations in the water phase were low (between 0.1 and 0.6 mg l^–1).     

Some effects of dibutylchloromethyltin chloride and other reagents on mitochondrial K+ flux

Respiration-dependent K⁺ fluxes across the limiting membranes of isolated rat liver mitochondria, measured by means of⁴²K, are stimulated by the oxidative phosphorylation inhibitor dibutylchloromethyltin chloride (DBCT). A lack of effect of Cl^– concentration indicates that the stimulation of K⁺ flux by DBCT is not attributable to Cl^–/OH^– exchange activity. The mercurial mersalyl was previously shown to stimulate respiration-dependent K⁺ influx. The combined presence of mersalyl plus DBCT results in a greater stimulation of K⁺ influx than is caused by either DBCT or mersalyl alone. The oxidative phosphorylation inhibitor oligomycin, which alone has no effect on respiration-dependent K⁺ influx, enhances the stimulatory effect of mersalyl on K⁺ influx. The data are consistent with, although not proof of, a direct interaction of the K⁺ transport mechanism with the mitochondrial energy transduction apparatus.Abbreviations used: DCCD,N,N-dicyclohexylcarbodiimide; DBCT, dibutylchloromethyltin chloride.     

Synchronous intra-Golgi transport induces the release of Ca from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca²⁺) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca²⁺ concentrations in the cell cytosol ([Ca²⁺]_cyt) and inside the lumen of the Golgi apparatus ([Ca²⁺]_GA), we have revealed transient increases in [Ca²⁺]_cyt during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca²⁺]_GA restoration ability. Thus, this redistribution of Ca²⁺ from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca²⁺-dependent phase of SNARE-regulated fusion of Golgi compartments.     

Crosstalk between cellular morphology and calcium oscillation patterns Insights from a stochastic computer model

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca²⁺]₁) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca²⁺ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such ‘Ca ²⁺ fingerprints’ can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca ²⁺ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca²⁺-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca²⁺ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca²⁺ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

In vitro synthesis and secretion of glycoprotein by human mammary tissue

Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([³H]glucosamine, [³H]fucose, [³H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [³H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.