The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.     

3H]tryptophan-[14C]proline dual label method for the simultaneous determination of collagen and noncollagen protein production

A new method for the simultaneous determination of newly synthesized collagen and noncollagen proteins has been developed. Because tryptophan is not found in collagen noncollagen proteins were specifically labeled with [3H]tryptophan. [14C]Proline was used to label both groups of proteins. To calculate the 14C-labeled noncollagen protein the 3H radioactivity of the protein mixture was divided by the ratio of 3H:14C in noncollagen protein of a representative sample. This value was obtained by collagenase digestion. The remaining 14C radioactivity in the protein mixture was attributed to [14C]collagen. There was a very good correlation between the dual label method and the widely used collagenase digestion method for the measurement of collagen and noncollagen protein production and for the calculation of the relative rate of collagen synthesis. This new method provides a simple and accurate analysis of collagen production, and it is suitable for rapid processing of a large number of biological samples.     

Mechanisms of DNA Utilization by Estuarine Microbial Populations

The mechanisms of utilization of DNA by estuarine microbial populations were investigated by competition experiments and DNA uptake studies. Deoxyribonucleoside monophosphates, thymidine, thymine, and RNA all competed with the uptake of radioactivity from [³H]DNA in 4-h incubations. In 15-min incubations, deoxyribonucleoside monophosphates had no effect or stimulated [³H]DNA binding, depending on the concentration. The uptake of radioactivity from [³H]DNA resulted in little accumulation of trichloroacetic acid-soluble intracellular radioactivity and was inhibited by the DNA synthesis inhibitor novobiocin. Molecular fractionation studies indicated that some radioactivity from [³H]DNA appeared in the RNA (10 and 30% at 4 and 24 h, respectively) and protein (approximately 3%) fractions. The ability of estuarine microbial assemblages to transport gene sequences was investigated by plasmid uptake studies, followed by molecular probing. Although plasmid DNA was detected on filters after filtration of plasmid-amended incubations, DNase treatment of filters removed this DNA, indicating that there was little transport of intact gene sequences. These observations led to the following model for DNA utilization by estuarine microbial populations. (i) DNA is rapidly bound to the cell surface and (ii) hydrolyzed by cell-associated and extracellular nonspecific nucleases. (iii) DNA hydrolysis products are transported, and (iv) the products are rapidly salvaged into nucleic acids, with little accumulation into intracellular nucleotide pools.     

Chromosome segregation in Escherichia coli B/r at various growth rates.

Chromosome segregation was analyzed in three substrains of Escherichia coli B/r growing at various rates. The cultures were pulse labeled with [14C]thymidine and bound to the bottom surface of a nitrocellulose membrane filter, and the radioactivity in newborn cells released from the surface during continuous elution with growth medium was measured. Since there was a fixed orientation in the release of newborn cells, the time course of the change in radioactivity per effluent cell could be used to investigate the orientation of chromosome segregation. If the radioactive deoxyribonucleic acid strands were partitioned at random between the progenies remaining attached to the membrane filter and those released into the effluent, the radioactivity per cell would decrease twofold after each generation of elution. The decrease in radioactivity was less than twofold at C + D min of elution and larger than twofold one generation later, indicating that chromosome segregation was nonrandom.     

Cleavage of adenosine 5''-monophosphate during uptake by Streptomyces griseus.

Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from [14C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [32P]AMP. Adenine inhibited uptake of [32P]AMP but not that of [32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of 32P and 14C from double-labeled AMP showed marked differences; 32P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide.     

Uptake and retention of external solutes from the digest medium during preparation of protoplasts

The extent to which solutes present in the digest medium enter cells and are retained during preparation of protoplasts was investigated. When barley (Hordeum vulgare, L. cv. Clipper) leaf slices were incubated in sorbitol there was considerable uptake of sorbitol into the tissue, which continued for up to 6 h and was dependent on the sorbitol concentration in the external medium. Protoplasts prepared by digesting leaf slices in a medium containing [¹⁴C]sorbitol but isolated and purified in media with unlabelled sorbitol contained significant amounts of [¹⁴C]sorbitol. From measurements of the protoplast volume, the internal sorbitol concentration was calculated to be 100 mM, assuming uniform distribution of the sorbitol throughout the protoplasm. The uptake of sorbitol during digestion and its retention by protoplasts was confirmed by measuring sugars in protoplast extracts by gas sucrose or inositol. Vacuoles prepared from the protoplasts contained 83% of the sorbitol present in protoplasts. It is concluded that considerable uptake of solutes from the external medium occurs during digestion of leaf tissue and that these solutes are retained within the protoplasts during isolation and purification. The solutes appear to be uniformly distributed throughout the subcellular compartments of the protoplast.     

Uptake of benzyladenine by excised watermelon cotyledons

The uptake of 8-[¹⁴C]N⁶-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.     

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies

The association of ¹²⁵I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of ¹²⁵I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation released a greater amount of rapid-dissociating radioactive label. Control experiments of [³H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the ¹²⁵I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of ¹²⁵I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells.     

Field diagnosis of urinary schistosomiasis by multiple use of nuclepore urine filters

It has been recognized recently that the standard field technique for the diagnosis of urinary schistosomiasis, urine filtration using reusable polyamide mesh filters, may give false-positive findings because filters cannot be washed adequately in all circumstances. In this study the alternative filtration method using polycarbonate membrane filters was tested, and the same problem existed. A variety of more vigorous washing procedures was field tested with the conclusion that polycarbonate filters can be washed adequately for reuse by a simple procedure that includes boiling for 5 min in tap water prior to washing with detergent.     

Filter types, filtration and post-filtration treatment in phytoplankton production studies

Acid rinsing, used to decontaminate filters in ¹⁴C productionstudies, caused cell rupture and resulted in elevated ¹⁴C countsin the filtrates of six out of seven phytoplankton samples.Large volume (50 ml) rinses using only water caused some, butlesser, damage. Comparing the recovery of ¹⁴C-labelled cellsand chlorophyll a on glass-fibre, polycarbonate and celluloseester filters revealed unaccountable losses at times with allthree filter types. These losses could not be explained by cellrupture, attachment to the filter funnel wall, filter treatmentor self-absorption during scintillation counting. Compared tothe whole sample acid bubbling method, recovery on the glass-fibrefilters was highest. Results for the polycarbonate filters weremore variable, while, in all cases, recovery on cellulose esterfilters was much lower.     

Effect of papaverine on the absorption and metabolism of 14C- adenosine and 14C-adenine in thymocytes

Some peculiarities of adenosine and adenine nucleotide metabolism in rat thymocytes were investigated. It was shown that the uptake of labelled adenosine or adenine by thymocytes is markedly inhibited by papaverine due to the decrease of the adenylate kinase activity, on the one hand, and to the acceleration of ATP catabolism and inosine and hypoxanthine release into the environment, on the other. ATP catabolism occurs in a special compartment which in [14C] adenosine and [14C] adenine prelabelled thymocytes has a higher specific radioactivity as compared with the whole cell. In [14C] adenine-prelabelled thymocytes and extracellular medium, papaverine does not influence the content but increases the specific radioactivity of adenosine.     

Filter assay technique and quench-flow experiments: examples of receptor-mediated transmembrane ion-exchange measured with membrane vesicles.

Modifications to a quench-flow apparatus are described which allow a rapid, in-line filter assay with immediate washing, in conditions to give minimum background. A design for an effluent spout is presented, which decelerates the liquid by a large factor, prevents splashes, limits the area of the filter exposed to the sample and allows an immediate wash over a larger area. A design for a filter assay funnel for general use is also presented. These devices feature minimal contact of the funnels with the filter disc. Examples are given in which in-line filtration was used to follow transmembrane ion flux in membrane vesicle preparations. In measurements of transmembrane flux with membrane vesicles and radioisotope the filter assay background can be resolved into three components. These are, (1) the uptake of radioactivity by the filter, (2) the radioactivity inside the vesicles not taking part in the specific measurement and (3) the occlusion of radioactivity in aggregated membrane particles on the filter. These different components depend on the conditions in different ways. Techniques for minimizing the background in filter assays are discussed. The importance of rapid filtration and immediate washing is demonstrated. The examples given illustrate that the function of the acetylcholine receptor from E. electricus is not affected by diisopropylfluorophosphate in the conditions used, and that added GABA is not removed from solution in a brain membrane preparation by the GABA uptake mechanisms in the short times of the experiments.     

Altered lipid synthesis in type II pneumonocytes exposed to lung surfactant.

When type II pneumonocytes were exposed to purified lung surfactant that contained 1-palmitoyl-2-[3H]palmitoyl-glycero-3-phosphocholine, radiolabelled surfactant was apparently taken up by the cells since it could not be removed by either repeated washing or exchange with non-radiolabelled surfactant, but was released when the cells were lysed. After 4 h of exposure to [3H]surfactant, more than half of the 3H within cells remained in disaturated phosphatidylcholine. Incorporation of [3H]choline, [14C]palmitate and [14C]acetate into glycerophospholipids was decreased in type II cells exposed to surfactant and this inhibition, like surfactant uptake, was half-maximal when the extracellular concentration of surfactant was approx. 0.1 mumol of lipid P/ml. Inhibition of incorporation of radiolabelled precursors by surfactant occurred rapidly and reversibly and was not due solely to dilution of the specific radioactivity of intracellular precursors. Activity of dihydroxyacetone-phosphate acyltransferase, but not glycerol-3-phosphate acyltransferase, was decreased in type II cells exposed to surfactant and this was reflected by a decrease in the 14C/3H ratio of total lipids synthesized when cells incubated with [U-14C]glycerol and [2-3H]glycerol were exposed to surfactant. Phosphatidylcholine, phosphatidylglycerol and cholesterol, either individually or mixed in the molar ratio found in surfactant, did not mimic purified surfactant in the inhibition of glycerophospholipid synthesis. In contrast, an apoprotein fraction isolated from surfactant inhibited greatly the incorporation of [3H]choline into lipids and this inhibitory activity was labile to heat and to trypsin. It is concluded that the apparent uptake of surfactant by type II cells in vitro is accompanied by an inhibition of glycerophospholipid synthesis via a mechanism that involves a surfactant apoprotein.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

Characteristics of Sorbitol Uptake in Rat Glial Primary Cultures

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.     

Evidence that the decarboxylation reaction occurs before the first methylation in ubiquinone biosynthesis in rat liver mitochondria

Biosynthesis of ubiquinone-9 was studied by incubating rat liver mitochondria with p-hydroxy[U-14C]benzoate, solanesyl diphosphate and S-adenosyl-L-methionine. When methylation reactions were inhibited by replacing S-adenosyl-L-methionine with S-adenosyl-L-homocysteine, nonaprenyl p-hydroxybenzoate and three other labeled peaks, designated as P1, P2 and P3 according to their retention times on HPLC, were observed. No carboxyl group was present in P1, P2 or P3 because the radioactivities disappeared when p-hydroxy[U-14C]benzoate was replaced by p-hydroxy[carboxyl-14C]benzoate. Compound P2 seemed to be hydroxylated but not methylated because its radioactivity markedly diminished under anaerobic conditions and the radioactivity was not incorporated into the compound from S-adenosyl-L-[methyl-3H]methionine, suggesting that P2 is 6-hydroxynonaprenylphenol. The complete correspondence of the retention times of P2 and chemically synthesized 6-hydroxynonaprenylphenol on HPLC further confirmed this possibility. P2 was a precursor of ubiquinone-9 because the radioactivity of the compound was incorporated into ubiquinone when incubated with mitochondria. The results suggest that the decarboxylation may occur prior to the first methylation in the ubiquinone biosynthesis in rat liver mitochondria, though it has been generally considered that in eukaryotes the first methylation precedes the decarboxylation.     

The affinity of [3H]quinuclidinyl benzilate and some other radioligands for glass microfiber and cellulose filters in some common liquid scintillation solvents

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.     

An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).     

Use of phosph-cellulose paper disks for the assay of histone acetyltransferase.

A modified method for the assay of histone acetyltransferase is presented. Previously reported methods depended upon the determination of the incorporation of radioactivity from [¹⁴C]acetyl coenzyme A into trichloroacetic acid (TCA)-precipitable material. However, as shown in this paper, [¹⁴C]acetylated histone cannot be precipitated quantitatively by TCA. In the method described in this paper, phospho-cellulose (P-cellulose) paper disks are used as an adsorbent for [¹⁴C]acetylated histone and 0.05 m carbonate buffer, pH 9.2, is used as a washing medium. This P-cellulose disk method allows more quantitative determination of [¹⁴C]acetylated histone than the TCA-precipitation methods.