Diffusion studies of bile acids,fatty acids and sucrose-NaCl-water systems at 37 °C by a modified capillary cell apparatus and their application to membrane transport studies

A capillary cell apparatus is described that allows accurate measurement of solute tracer diffusion coefficients in biological solutions at 37 °C. The apparatus has a unique stirring mechanism to provide a uniform flow pattern over the capillaries with only 18 ml of the bulk solution. Four capillaries of 2 cm length are used. With this apparatus measurement can be made at relatively short time periods so that bacterial overgrowth in the solutions is minimized. Using this apparatus tracer diffusion coefficients of three bile acids, cholic, taurochollic and taurodeoxycholic acids, and four fatty acids, acetic, pentanoic, octanoic and decanoic acids, were measured in an isotonic phosphate buffer, pH 7.1, at 37 °C. Viscosity, density and diffusion coefficients of sucrose in physiological saline solutions were also measured.     

The mechanics of pulling a glass micropipette.

The pulling of micropipette electrodes from glass tubing has been treated as a problem of viscous flow coupled with the Newtonian dynamics of the pulling apparatus. Analytical solutions are given from which the taper profile, tip diameter, and pulling time can be obtained. The physical principles of operation of micropipette pullers are discussed.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

Nutation in Seedling Phaseolus Multiflorus

A simple apparatus is described which enables records to bemade of the nutational movements of small amplitude exhibitedby seedling plants. The apparatus described has a resolutionof o.1 mm and 5 minutes. The recording system is capable ofrecording over periods of up to 24 hours' duration automatically. Using this apparatus, numerous recordings have been made ofthe nutation of runner-bean seedlings. The movement exhibitedis considered to be the result of a periodic, oscillatory movement,namely nutation, upon which are superimposed linear movementscaused by tropic responses or random growth irregularities.The apparatus has been found especially useful for the studyof the periodicity of nutation, and a study of the variabilityof this parameter in the nutation of the bean seedling at aconstant temperature has been made as an essential prerequisitefor the study of the effect of experimental treatments. Thevariability has been shown to be high, even within the sameplant.     

An airlift pump device for low pressure perfusion storage of the isolated heart

A portable apparatus for the continuous hypothermic perfusion of the isolated heart is described. The system has been used successfully to store pig and baboon hearts for periods of up to 48 hr, and to store human donor hearts for periods of 7 to 17 hr before being transplanted. The perfusate is both oxygenated and circulated by gas flow from a pressurized oxygen cylinder, using the air-lift pump principle. The apparatus has no moving parts and requires no electrical energy supply; malfunction is, therefore, extremely unlikely. A regulator has been incorporated which can be adjusted to increase or decrease the myocardial perfusion pressure. The system and environmental variables which can affect flow and pressure within the apparatus are discussed. The storage time allowed by this system will enable transportation of donor hearts between most of the world's major cities.     

An automatic apparatus for continuous suspension culture with a concentrating device

A chemostat in which mammalian cells can be raised in continuous suspension culture is described. It is constructed from commercially available parts. This apparatus has the advantage over earlier models in that the medium can be pumped off free of cells, thus suddenly increasing the cell concentration in the culture. The apparatus has been successfully used in studies on contact inhibition.     

Purification and concentration of albumin solutions by diafiltration using a parallel membrane dialyzer]

The last two years, artificial Kidneys have been used for purification and concentration of human serum albumin solutions coming from plasma cracking in two Blood Transfusion Centers. Results are easily reproducible and the apparatus is reliable and of low cost. The properties of dialysis and ultrafiltration of the A.N. 69 membrane are useful for eliminating ethanol and water. The artificial Kidneys are effective at low pressure. It is then possible to use peristaltic pumps and to have a closed circuit. The whole apparatus must be sterilized with chemical reagents.     

A procedure for the simultaneous determination of small quantities of hyaluronate and isomeric chondroitin sulfates by chondroitinases

A simple, commercially available apparatus is described which can be used for injection of nanoliter quantities of aqueous solutions into the germinal vesicle or the cytoplasm of Xenopus laevis oocytes.     

Use of a glass electrode for measuring rapid changes in photosynthetic rates

1. A continuously recording glass electrode apparatus has been described for measuring carbon dioxide concentration changes in solution. The limits of applicability of the apparatus have been analyzed. 2. The glass electrode apparatus has been used for the measurement of transient rates of photosynthesis by algal suspensions. 3. The decline of the photosynthetic rate in high light at carbon dioxide partial pressures less than 0.5 per cent atmosphere, observed in the glass electrode apparatus, has been confirmed by steady state experiments in which flowing gas streams were analyzed.     

STRUCTURE AND SIGNIFICANCE OF THE CRYPTOMONAD FLAGELLAR APPARATUS. I. CRYPTOMONAS OVATA (CRYPTOPHYTA)1

The absolute configuration of the flagellar apparatus in Cryptomonas ovata has been elucidated and found to be similar to that reported for Chilomonas paramecium. Variations apparent in the flagellar apparatus of Cryptomonas ovata include the presence of striations in the mitochondrion associated lamella, a rhizostyle which does not bear wing-like extensions from the microtubules and does not lie close to the nucleus, and a striated fibrous anchoring structure associated with one basal body which has not hitherto been described. The flagellar apparatus also includes a four stranded microtubular root which traverses into the anterior dorsal lobe of the cell, a striated fibrous root which is associated with a five stranded microtubular root, and a two stranded Cr root. The homologous nature of these roots to those in the larger cryptomonads is discussed in relation to the apparent reduction in flagellar apparatus size and complexity among the smaller cryptomonads. A diagrammatic reconstruction of the flagellar apparatus of Cryptomonas ovata is also presented.     

A GLASS ELECTRODE APPARATUS FOR MEASURING THE pH VALUES OF VERY SMALL VOLUMES OF SOLUTION

A glass electrode apparatus is described with which pH measurements can be made with as small volumes as 2 drops (about 0.14 cc.) of solution. Using this apparatus the change of pH of the vacuolar sap of Nitella, due to the penetration of brilliant cresyl blue, has been readily followed. The sap and the dye have been found to poison the usual type of hydrogen electrode.     

The response of bacterial spores to vacuum treatments. I. Design and characterization of the vacuum apparatus

A vacuum apparatus has been described that has enabled samples of bacterial spore suspensions to be dehydrated at defined temperatures between 0 and 65 °C, with facilities for reequilibration of the dried samples to aqueous vapor pressures between 5 × 10^?4 and 10 torr and subsequent exposure to dry gases. The apparatus has been characterized using sample temperature/drying-time profiles, and drying rate/ drying-time curves, and the reproducibility of the dehydration and rehydration techniques has been established. Biological data have confirmed the suitability of the apparatus since no loss of spores from samples has been observed during any of these experimental treatments. On the basis of measurements recorded during rehydration of dried spores, it is suggested that dehydration occurs at specific sites in the spore which are of two types, 1) reversibly dehydrated (rehydratable) and 2) irreversibly dehydrated (nonrehydratable).     

ISOLATION OF THE GOLGI APPARATUS FROM PLANT CELLS

A method for the isolation of the Golgi apparatus from stem tissues of onion is described. Preparations that consisted mainly of morphologically identifiable Golgi apparatus have been obtained. The best preparations were obtained from tissue homogenized under conditions of minimum shear, and in the presence of sucrose and certain additives which aid in preservation of the integrity of the Golgi membranes. Those additives, which had a pronounced stabilizing effect on the isolated apparatus, included both monovalent and divalent ions (sodium and calcium) and dextran. A large portion of the Golgi apparatus did not appear to change microscopic appearance upon isolation, but were observed to fuse into large aggregate structures not unlike those occurring naturally in certain animal or insect cells (12). Fusion occurred both at the edges of the cisternae and in register, but the integrity of the individual cisternae was not destroyed. The major contaminants of the Golgi apparatus fraction were numerous small and large spherical vesicles. At least some of these vesicles appeared to have been derived from the Golgi apparatus; others may have been fragments of the cell membrane, the endoplasmic reticulum, or other cell debris. By utilizing this procedure, it has been possible to obtain fractions of Golgi apparatus from plant tissues other than onion stem. However, at the present time it is only with onion that the Golgi apparatus has been isolated in a form that would warrant further purification for biochemical analysis.     

Physical membrane displacement: Reconstitution in a cell-free system and relationship to cell growth+

Summary Physical membrane displacement is a process common to all forms of vesicle budding as well as cell enlargement and pleomorphic shape changes. Cell-free reconstitution of membrane budding has been achieved with transitional endoplasmic reticulum fractions from both plants and animals where 50 to 70 nm transition vesicles have been observed to bud from the part-rough, part-smooth membrane elements that define transitional endoplasmic reticulum. This budding phenomenon requires ATP, is facilitated by cytosol and guanine nucleotides, and is both time- and temperature-dependent. The transitional endoplasmic reticulum buds that form when concentrated by preparative free-flow electrophoresis will attach specifically to cis Golgi apparatus membranes immobilized on nitrocellulose as an acceptor compartment. Golgi apparatus membranes derived from the trans compartment do not serve as an efficient acceptor compartment. Transfer of the vesicles once formed is rapid, nearly complete and no longer dependent upon added ATP. Transfer shows a strict temperature dependency corresponding to that of the intact cell where at temperatures of 16°C or below, vesicles form but do not attach to cis Golgi whereas at temperatures of greater than 16°C, vesicles both form and fuse. The principle ATPase of transitional endoplasmic reticulum which may be involved in the budding process has been identified, characterized and isolated. A 38 kDa cis Golgi apparatus associated protein also has been identified as a potential candidate as a docking protein. Transfer between trans Golgi apparatus and the plasma membrane also has been studied by cell-free analysis. Here, transfer has been found to be stimulated by NADH or NADH plus ascorbate. The role of NADH is unknown but the ability of plant and Golgi apparatus to oxidize NADH is inhibited by brefeldin A, a compound known to block membrane trafficking even at the level of the trans Golgi network. NADH oxidase activity of plasma membranes also has been described and is inhibited as well by brefeldin. Recent observations suggest that brefeldin A may block both the formation of vesicles at the trans Golgi apparatus as well as auxin hormone-stimulated cell elongation in plants. This once again raises the possibility of whether or not plant cell elongation is obligatorily mediated by membrane input from the Golgi apparatus. The latter seems unlikely based on two additional lines of evidence. The first is that auxin-induced cell elongation in plants shows no sharp temperature transition over the range of 4 to 24°C, whereas production of secretory vesicles from the trans Golgi apparatus appears to be largely prevented at temperatures of 18°C or less. Secondly, the sodium selective ionophore, monensin, which effectively blocks the formation of functional secretory vesicles at the trans Golgi apparatus, is also largely without effect on auxin-induced cell elongation for periods of 4 h or longer. Taken together the findings suggest that the action of brefeldin A on vesicle budding at the Golgi apparatus and cell enlargement, are not directly correlated but may represent a common action of the drug on some constituent essential to membrane displacement mechanisms.Abbreviations BFA brefeldin A - IAA indole-3-acetic acid; 2, 4-D 2, 4-dichlorophenoxyacetic acid - NSF N-ethylmaleimide-sensitive factor Much of the information summarized in this report was presented as a plenary lecture at the XV International Botanical Congress Tokyo, Yokohama, Japan, August 28–September 3, 1993.     

A quenched-flow apparatus which allows the measurement of the kinetics of a reaction in one stroke

A chemical quenched-flow apparatus is described which measures, in a unique stroke, enough data points (8–11) for establishing the kinetics curve of a reaction. Only very small volumes of reaction solutions (2 × 500 μl) are required. The time intervals between which the kinetic data may be measured range from 5 to 37 ms and from 120 to 450 ms with the corresponding mixing times of 0.6 and 5 ms, respectively. This apparatus was used to investigate the pre-steady-state domain of the aminoacylation reaction of tRNA^Val by valyl-tRNA synthetase from yeast.     

A simple and inexpensive apparatus for measuring fish metabolism

A simple, noninvasive, and economical home-made respirometer has been used to determine the standard metabolism of goldfish. The apparatus has been tested on several fishes and has proved its accuracy in determining a mass effect on standard metabolism. The apparatus can be made easily by middle school, high school, and undergraduate students and can be used to introduce them to basic concepts in animal physiology and in biological statistics.     

Theory for the design of apparatus for drying frozen tissues

A general theory for the design of apparatus for the low temperature drying of frozen tissues has been developed. Using this theory it is possible to compute drying rates for different materials in different apparatuses and to design an apparatus suitable for the required purpose. It is shown that for the drying of animal tissues in the laboratory, where the area is small, the requirements of the apparatus are not particularly critical. The same theory applied to the drying of biologicals and plasma indicates that the design is exceedingly critical and suggests directions in which existing apparatus might be modified. Aided by a grant from the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago and from the Commonwealth Fund of New York.     

OBSERVATIONS ON THE RELATIONSHIP OF THE GOLGI APPARATUS TO WALL FORMATION IN THE MARINE CHRYSOPHYCEAN ALGA, PLEUROCHRYSIS SCHERFFELII PRINGSHEIM

The role of the Golgi apparatus in wall formation of vegetative cells of a marine chrysophyte, Pleurochrysis scherffelii, is described. Wall fragments are synthesized within the cisternae of the Golgi apparatus. A single Golgi apparatus is always located at the cell periphery, and the distended cisternae are oriented toward the cell surface. A highly-ordered body found near the inflated cisternae is associated with spherical, membrane-bounded bodies which may be involved in the progressive degeneration of cisternal membranes which release wall fragments. Protoplast movement has been detected by time-lapse cinephotomicrography and is correlated at the ultrastructural level with change in positions of the Golgi cisternae. Wall-synthesizing capacity is greatest during transverse wall formation. Senescent cells lack a Golgi apparatus with inflated cisternae. In addition, wall fragments are not present in the Golgi cisternae at this stage. Zoosporogenesis results in a temporary loss of the wall-forming capacity of the Golgi apparatus; this activity then resumes with the formation of a different morphological entity, the scale. Preliminary quantitative measurements of the turnover capacity of the Golgi apparatus have been made. From these data it has been determined that between 41 and 82 Golgi generations are required to synthesize the cell wall of an actively growing cell; this estimate indicates that approximately one cisterna is produced every 2 min, provided the cell generation time is 3 days. The time-lapse cinephotomicrographic data confirm that the rate of production of Golgi cisternae is at least one cisterna every 2 min.     

A simple preparative polyacrylamide disc gel electrophoresis apparatus: purification of three branched-chain amino acid binding proteins from Escherichia coli

The equipment used for preparative polyacrylamide gel electrophoresis has been either difficult to construct or costly if purchased commercially. An inexpensive preparative acrylamide gel apparatus and peristaltic pump are described in this paper which are easy to use and may be constructed from readily available materials. The construction of the preparative gel apparatus requires no special machining or glass blowing.This report describes the use of the disc gel apparatus in the final purification step of three binding proteins which appear to be involved in the transport of the branched-chain amino acids in Escherichia coli. Two of these proteins have been described previously (1–4). The apparatus has also been successfully used in a number of other laboratories for the purification of a variety of other proteins (5–9).     

Ultrasonification of biological materials. I. Apparatus

A self-aligning apparatus for use with a sonifier is described that permits reproducible ultrasonic treatment of solutions. The apparatus also reduces exposure of personnel to high-intensity sound.