THE PREPARATION OF THE GRADED COLLODION MEMBRANES OF ELFORD AND THEIR USE IN THE STUDY OF FILTERABLE VIRUSES

1. The method described by Elford for the preparation of graded collodion membranes suitable for ultrafiltration was found to give excellent results, and his findings are fully confirmed. 2. A formula is given for the preparation of collodion from which satisfactory membranes of graded porosity can be prepared. 3. The technique and apparatus used in the preparation, and standardization of membranes are described in detail. 4. The technique and apparatus required for ultrafiltration experiments are described, and some drawbacks encountered in the experiments are discussed. 5. The results of ultrafiltration experiments show that the pores of the membranes are remarkably uniform in size.     

A new, rapid, method for preparation of dispersed pancreatic acini.

A new method for the preparation of pancreatic acini is described. The method is simple and much more rapid than previously described techniques, the time required for preparation of pancreatic acini being 20 min from removal of the pancreas. Acini prepared with this method perform in a superior manner when stimulated by either caerulein or secretin. Thus this new technique would be ideal for use in binding and secretion studies.     

A simplified method for the rapid preparation of peroxidase-anti-peroxidase (PAP) complexes

Summary A modified method is described for the rapid production of peroxidase-antiperoxidase complexes to be used in immunocytochemistry. In this method anti-peroxidase antibodies are precipitated from crude serum with peroxidase at equivalence and subsequently resolubilized at low pH with excess peroxidase. The complexes are isolated from unbound immunoglobulin and peroxidase by gelfiltration. The method combines the advantages of both previously described preparation procedures. The resulting PAP-complex, when tested in indirect immunocytochemistry, is comparable to that obtained in established preparation procedures.Supported by a grant from the Queen Wilhelmina Cancer Foundation (grant no. PA 77/52)     

Transport of catecholamines by resealed chromaffin-granule `ghosts''

A method is described for the preparation of resealed chromaffin-granule ;ghosts'. The lysis and rapid purification procedures provide ;ghosts' in approximately 70% yield from crude granules; the preparation contains 0.1mumol of catecholamine/mg of protein (as compared with 2.8mumol/mg in unlysed granules), of which about one third is inside the ;ghosts'. The ;ghosts' retain their ability to accumulate catecholamines, a process dependent on Mg-ATP and inhibited by reserpine, and a simple assay for this transport is described.     

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Preparation of arylzinc reagents and their use in the rhodium-catalyzed asymmetric 1,4-addition for the synthesis of 2-aryl-4-piperidones

A protocol for the catalytic asymmetric synthesis of 2-aryl-4-piperidones with high enantiomeric excess (ee) (typically > or = 99% ee) has been described here. The preparation of arylzinc reagents, which are used as nucleophiles in catalysis, is also included. The whole protocol can be completed in 10-20 h, starting from the preparation of the arylzinc reagents, depending on the reaction time of the rhodium-catalyzed process. A detailed protocol is described using the preparation of 4-fluorophenylzinc chloride and its addition to benzyl 3,4-dihydro-4-oxo-1(2H)-pyridinecarboxylate in the presence of [RhCl((R)-binap)]2 as an example.     

The rapid preparation of peroxisomes from rat liver by using a vertical rotor.

A method is described for the rapid preparation of peroxisomes from rat liver by using sucrose-density-gradient centrifugation in a vertical rotor. The preparation, shown to be virtually free of mitochondrial and microsomal contamination, can be used to study fatty acid metabolism by isolated peroxisomes.     

Purification of brush border membrane vesicles from horse kidney cortex using Percoll

A rapid method for preparation of brush border membrane vesicles from a large amount of horse kidney cortex is described. Self-orienting Percoll-gradient centrifugation minimized contamination by microsomal membranes. The characteristics of this preparation were checked by electron microscopy and measurement of L-alanine uptake.     

Preparative scale isolation of sphingosine

A high-yield method is described for the preparation of sphingenine from galactosylceramide. Two successive cleavage reactions are used, alkaline butanol to form galactosylsphingosine, then acidic acetonitrile to form the sphingenine. A column purification procedure utilizing silica gel is described. The method is superior to previously described methods in its rapidity and avoidance of isomerization and contamination by racemization and rearrangement products.     

Preparation of electrophoretically homogeneous erythrocuprein and its thermodenaturation]

The procedure for isolation of electrophoretically homogeneous preparation of erythrocuprein from 17 1 blood batch was described. The preparation had absorbtion value, A260/A680, of 25-26. The protein completely retains superoxide dismutase activity after treatment 7 min. at 75 degrees C. The heating of protein solution at 100 degrees C bring about autoreduction of protein copper as demonstrated by decreasing optical and EPR-spectral intensities. The reduced protein was oxidized in aerobic condition and oxidized preparation had optical and EPR-spectra differ from those of the native protein the activy being decreased.     

Medium-Scale Production and Purification of Ochratoxin A, a Metabolite of Aspergillus ochraceus

The preparation of crystalline ochratoxin A from Aspergillus ochraceus nutrient solution is described. Methods are adaptable to large-scale fermentations.     

Purification of microvillus membrane vesicles from pig small intestine by adsorption chromatography on sepharose

A simple, rapid method for the preparation of pure microvillus membrane vesicles from pig small intestine is described. The method is based on the ability of agarose beads to adsorb selectively the impurities, mainly basolateral membrane fragments, from a microvillus vesicle preparation isolated by hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

A procedure for the rapid preparation of mitochondria from rat liver.

A technique for the rapid preparation of mitochondria from rat liver is described. Tissue fractionation is performed by a single centrifugation step with a discontinuous Percoll density gradient. Total preparation times of 5--6 min are achieved by using this method. The mitochondrial fraction obtained is relatively free of contaminating organelles, as judged by marker-enzyme activity determinations. Mitochondria isolated by Percoll-density-gradient centrifugation differ from mitochondria obtained by differential centrifugation [Taylor, Prpić, Exton & Bygrave (1980) Biochem. J. 188, 443--450] in that the former exhibit a higher acceptor control ratio and a higher calcium content. Values obtained for the protonmotive force are not significantly different between the two preparations. The technique described may be widely applicable for studies requiring the rapid preparation of functionally intact and relatively uncontaminated mitochondria.     

Gel filtration of myelin using 2,2,2-trichloroethanol as the solvent.

The use of 2,2,2-trichloroethanol as a solvent for myelin from both the central and peripheral nervous systems is described. Concentrated, optically clear solutions of lyophilized myelin in this solvent are stable for weeks. The preparation of highly concentrated myelin proteins by gel filtration in trichloroethanol is described.     

Agarose-acrylamide gradient gel electrophoresis of proteins

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.     

Amino acid pharmacology in a mature rat spinal cord preparation in vitro

1. It has been shown that a relatively large intact piece of mature mammalian spinal cord can be maintained in vitro if suitable experimental conditions are employed. 2. The preparation as described gives robust and reproduceable reflex responses in both dorsal and ventral horns (ventral horn activity has been maintained for over 36 hr in vitro). 3. The action of antagonists at both excitatory and inhibitory amino acid receptors give qualitatively predictable results from known in vivo experiments with the added advantage of fine quantitative control. 4. A significant advantage of such an adult preparation over more common immature spinal cord preparation is the ability to clearly separate low threshold reflexes, such as those described in this article, from reflexes evoked by small diameter non-myelinated afferents.     

The purification and properties of extracellular glycosidases of the cellular slime mould Dictyostelium discoideum

The purification of beta-N-acetylhexosaminidase, alpha-glucosidase, alpha-mannosidase and beta-glucosidase from the spent growth medium of Dictyostelium discoideum strain Ax-2 myxamoebae is described. beta-N-Acetylhexosaminidase and alpha-glucosidase were obtained in high yield and as homogeneous preparations whereas the alpha-mannosidase preparation consisted of two electrophoretically distinct isoenzymes. The physical, chemical and kinetic properties of these enzymes are described.     

Spontaneous fluctuations in the activity of rabbit skeletal muscle NAD-kinase]

Spontaneous fluctuations in the time of the activity of the 280-300-fold purified NAD-kinase preparation from rabbit skeletal muscle following its dilution are described. Defrosted but undiluted enzyme preparation failed to exhibit any fluctuations in its activity.     

Gel filtration of myelin using 2,2,2-trichloroethanol as the solvent

The use of 2, 2, 2-trichloroethanol as a solvent for myelin from both the central and peripheral nervous systems is described. Concentrated, optically clear solutions of lyophilized myelin in this solvent are stable for weeks. The preparation of highly concentrated myelin proteins by gel filtration in trichloroethanol is described.