Reverse phase high-pressure liquid chromatographic separation of retinoids,including retinylphosphate and mannosylretinylphosphate

Reverse phase hplc was used to separate retinylphosphate mannosylretinylphosphate from other retinoids. These phosphorylated retinoids constituted 12 and 42% of the extracted radioactivity in OEL mouse intestine at 3 and 24 h respectively after the intubation of [1-³H]retinol. A detailed procedure for the chemical synthesis of retinylphosphate is described.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[¹⁴C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[¹⁴C]glucose.Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes.

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[14C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[14C]glucose. Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.     

A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria

Summary A procedure is described for the calculation of solute fluxes in mitochondria from absorbance measurements. The procedure assumes that mitochondria behave as osmometers and that they are always at osmotic equilibrium.The rates and amounts of K⁺ translocation have been calculated simultaneously, with the photometric procedure and electrometrically, during passive, K⁺ efflux coupled to Ca⁺⁺ uptake and during active K⁺ uptake and passive K⁺ release coupled with anion translocation. Good agreement has been found between the two sets of measurements. The data are compatible with the concept that the energy-linked, ion translocation-coupled, mitochondrial swelling is osmotic in nature. It is concluded that the changes of absorbance are quantitatively related to changes in the inner volume and therefore the photometric procedure can be used to calculate, ion fluxes of osmotically active species under various circumstances.     

The Isolation and Characterization of The Lamellar Body of Bovine Lung

A novel procedure for preparing intact lamellar bodies from bovine ³ lung has been developed. The procedure consists of enzymatic digestion ⁴ of ground-lung tissue, with Nagarse, homogenization with a loosely-fitting pestle and then differential and density gradient separation of the various cellular fractions as the basis for selection of the lamellar body fraction.     

Preparation of highly radioactive homogeneous 32P-labeled hydroxymethylglutaryl coenzyme A reductase from rat liver

A procedure for the preparation of highly radioactive homogeneous ³²P-labeled 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver microsomes has been developed. The enzymatic preparation obtained by this procedure has a specific radioactivity 50-fold higher than that reported in previous literature. The purified enzyme was judged to be homogeneous on the basis of comigration of enzyme activity with a single band of protein and ³²P radioactivity on polyacrylamide gels. The ³²P covalently bound to the reductase was removed upon incubation with purified hydroxymethylglutaryl coenzyme A reductase phosphatase from rat liver.     

Reconstitution of an ATP-dependent sodium pump with an ATPase from electric eel and pure phospholipids.

An ATP-dependent sodium pump was reconstituted with an ATPase preparation from the electric eel and purified phospholipids. Vesicles reconstituted by the sonication procedure catalyzed Na⁺ translocation at a rate 10 to 20 times higher than vesicles reconstituted by the cholate-dialysis procedure. A small but reproducible stimulation of Na⁺ transport by valinomycin in the presence of K⁺ was observed.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

The release of endonuclease I from Escherichia coli by a new cold shock procedure

A new cold shock procedure has been developed for releasing large quantities of endonuclease I from $\text{E. coli}$, which neither involves EDTA-lysozyme treatment nor osmotic shock. Treatment of cells with ice-cold 0.1M Tris-0.2M KCl buffer, pH 7.4 results in the release of endonuclease I into the medium. Although the loss of endonuclease I from the cells is a rapid process, its recovery in the shock fluid is gradual and approaches maximum in about 90 minutes. Certain divalent metal ions such as Mg⁺⁺ and Mn⁺⁺ strongly inhibit the release of endonuclease I. The cold shock procedure is rather selective and the mechanism of the release of endonuclease I is different from that of osmotic shock procedure.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

Sequential individual resonance assignments in the 1H-nmr spectra of polypeptides and proteins

Recently, a new procedure for the assignment of protein ¹H-nmr spectra was introduced that relies on stereochemical considerations of proton–proton distances in polypeptides and on the use of two-dimensional nmr for obtaining ¹H-¹H through-bond and through-space connectivity maps. In the present paper a particular aspect of this assignment procedure is discussed in more detail, i.e., how to obtain individual resonance assignments from identification of amino acid side-chain spin systems and identification of neighboring residues in the amino acid sequence.     

Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

A rapid isolation procedure of nucleoli from detergent purified nuclei of some tumor and tissue culture cells is described. The procedure makes use of a non-ionic detergent, Nonidet P40 and sodium deoxycholate to purify nuclei followed by the addition of Ca²⁺ or Mg²⁺ to strengthen the nucleoli against sonication. Enzymatically active (with respect to nucleolar RNA polymerase) nucleoli containing undegraded nucleolar RNAs may be isolated from a mouse hepatoma MH134, Ehrlich ascites tumor, HeLa cells, L cells and C3H2K cells with this procedure.     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

Elemental sulfur production during mixotrophic growth on hydrogen and thiosulfate of thermophilic hydrogen-oxidizing bacteria

In this study we measured the exogenous production and the intracellular content of elemental sulfur (S⁰) in the thermophilic sulfur-oxidizing bacteriaHydrogenobacter spp. andBacillus schlegelii during mixolithoautotrophic growth on hydrogen and thiosulfate. Under these conditions, all strains produced and released white-yellow hydrophilic S⁰ particles into the growth medium. Hydrophilic S⁰ was separated from cells by a differential low-speed centrifugation procedure. The S⁰ pellets were dried, and the S⁰ was purified by column chromatography and by thin-layer chromatography (TLC). The S⁰ TLC-band could be stained with triphenyltetrazolium chloride and piperidine procedure. Determination of intracellular S⁰ content was performed from fresh cells absolutely free of exogenous S⁰ particles. quantitation of S⁰ was performed by high-performance liquid chromatography, colorimetric thiocyanate procedure, and by UV-spectra analyses. All the strains studied, in particularB. schlegelii strains, released significant quantities of S⁰ into the growth media. In contrast, the intracellular S⁰ content was very low. Significant rhodanese activity in the presence of thiosulfate was measured with toluenepermeabilized cells and cell-free extracts ofB. schlegelii (type strain) andHydrogenobacter spp. (strain T3).     

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Determination of [d-Ala2, d-Leu5]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid

A routine procedure has been established for the sensitive and specific detection of avocado sunblotch viroid in partially purified nucleic acid extracts of avocado leaves by hybridisation analysis with ³²P-complementary DNA prepared against the purified viroid. Avocado sunblotch viroid was shown to be present in 12 avocado trees that had indexed positive in a biological test for sunblotch disease but was absent from 10 trees that indexed negative. The complete correlation between sunblotch disease and the presence of viroid indicates that the complementary DNA hybridisation assay procedure can be used for the indexing of sunblotch disease. The overall procedure of leaf extraction and hybridisation analysis can be completed in 5 days and is to be compared with up to 2 yr required for indexing by biological methods. The level of avocado sunblotch viroid in partially purified nucleic acid extracts of a number of different sources of sunblotch infected avocado leaves was found to vary 10 000-fold from 0.2% to 2 × 10^-5% by weight. The lower limit of detectability of the viroid by the hybridisation assay is considered to be about 10^-5% by weight; this is at least 10³ times more sensitive than the detection of the viroid by polyacrylamide gel electrophoresis of the leaf nucleic acid extracts followed by staining.     

Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [¹⁴C]δ-aminolevulinic acid with a high yield of conversion (?60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (?30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [¹⁴C]protoporphyrin preparation.     

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.