Release of hyaluronate from eukaryotic cells.

The mechanism of hyaluronate shedding from eukaryotic cell lines was analysed. All cell lines shed identical sizes of hyaluronate as were retained on the surface. They differed in the amount of hyaluronate synthesized and in the proportions of hyaluronate which were released and retained. A method was developed which could discriminate between shedding due to intramolecular degradation and that due to dissociation as intact macromolecules. This method was applied to B6 and SV3T3 cells in order to study the mechanism of hyaluronate release in more detail. The cells were pulse-labelled to form hyaluronate chains with labelled and unlabelled segments, and the sizes of labelled hyaluronate released into the medium during the pulse extension period were determined by gel filtration. B6 cells released identical sizes of hyaluronate at all labelled segment lengths, indicating that no intramolecular degradation occurred. When chain elongation was blocked by periodate-oxidized UDP-glucuronic acid, hyaluronate release was simultaneously inhibited. These results indicated that B6 cells dissociated hyaluronate as an intact macromolecule. In contrast, SV3T3 cells released hyaluronate of varying molecular mass distributions during extension of the labelled segment, suggesting partial degradation. Exogenous hyaluronate added to SV3T3 cultures was also degraded. This degradation could be prevented by the presence of radical scavengers such as superoxide dismutase and tocopherol. Degradation of endogenous hyaluronate could be inhibited by salicylate. These results led to the conclusion that SV3T3 cells released hyaluronate not only by dissociation, but also by radical-induced degradation.     

Solid phase synthesis of [18F]labelled peptides for positron emission tomography

A strategy for the solid phase synthesis of [18F]labelled peptides has been developed. The peptides were prepared on solid support and acylated with 4-[18F]fluorobenzoic acid using HATU within 3 min and the labelled peptide was released from the solid support within 7 min. The [18F]labelled peptides were produced in good purity with a specific activity of 20-25 GBq/micromol.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

Metabolism of Adenine in Healthy and Blighted Potato Leaves

Analysis of the distribution of ¹⁴C in extracts prepared fromleaf tissue which had been exposed to labelled adenine by petiolaruptake revealed that this purine is extensively metabolizedin both healthy and Phytophthora-infected potato leaves. Incorporationof labelled adenine into the major ribonucleic acid speciesof the leaf was also extensive as determined by radioactiveassays performed on individual fractions which were separatedon columns of methylated albumin kieselguhr. Examination ofindividual nucleotides released by alkaline hydrolysis showedthat both the adenylic and guanylic acid moieties were labelled.Although the labelling patterns were similar for RNA from healthyand infected leaf tissue, the specific activity of the latterwas consistently higher than the former. When partially purified leaf extracts were assayed for phosphoribosyltransferase,they exhibited relatively high levels of activity with adenineas substrate, but were virtually devoid of activity with hypoxanthineand guanine. However, direct petiolar uptake of labelled hypoxanthineresulted in highly labelled RNA. A comparison of adenine phosphoribosyltransferase activity inextracts from healthy and blighted leaves failed to reveal measurabledifferences. Therefore, it was concluded that the differentialincorporation of labelled adenine into the RNA of healthy andinfected leaves was due neither to increased activity of thisenzyme in response to infection nor to its differential activation. Apart from its role in the recovery of preformed purines fornucleic acid synthesis, adenine phosphoribosyltransferase mayfunction as part of a mechanism for regulating levels of adeninein the potato leaf.     

Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways.

The ability of human keratinocytes and squamous carcinoma cell lines to attach lipid covalently to cell proteins has been examined using both palmitic and myristic acids. SDS-polyacrylamide gel analyses of the proteins labelled with these lipids demonstrated that each labelled a different set of proteins. Covalently protein bound palmitic acid could be removed from the proteins by mild alkali hydrolysis but the bound myristic acid required prolonged acid hydrolysis to release it from the associated proteins. H.p.l.c. analyses of the released lipid confirmed that both lipids were attached to proteins directly and that the labelling was not due to the lipids being catabolised. Cycloheximide could prevent the attachment of myristic acid to cell proteins, but only reduced the levels of palmitic acid incorporation. Pulse chase experiments indicated that there was little turnover of the attached myristic acid whereas this was significant for covalently bound palmitic acid. These observations show for the first time that two different protein populations are labelled by different lipids in eukaryotic cells, and that there appear to be two separate pathways for the acylation of proteins in such cells.     

Cell surface sialoglycopeptide metabolism and surface glycosyl transferase activity in the Ehrlich ascites cell.

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained. 2. However, when intact cells were incubated with labelled uridine 5'-diphosphate-N-actely glucosamine or cytidine 5'-monophosphate (CMP)-sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity in the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP-sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP-sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.     

Identification of the lipids liberated by the isolated intestine from Periplaneta americana after ingestion of doubly labeled tripalmitine]

It has been shown by incubation of isolated midgut from Periplaneta americana fed double labelled triglycerides (3H glycerol, 14C oleic acid) that mostly triglycerides but also diglycerides and free fatty acids are released by the midgut. Fed triglycerides have been hydrolysed, then resynthetised before being released.     

Radioiodination of L 1210 cells.

The lymphoid leukaemia L 1210 cells of mice were labelled with 125I. The cell homogenates were fractionated and from the microsomal fraction 90 per cent of the radioactive material could be precipitated with perchloric acid, whereas only 4 per cent was precipitated from the soluble fraction. Papain bound with Enzacryl AH released 31 per cent of radioactivity. It was concluded therefrom that the surface proteins of the cells were labelled. Electrophoretic separation of these proteins in polyacrylamide gel with sodium dodecyl sulphate was performed and 6--8 radioactive fractions of surface peptides were found.     

A STUDY OF THE METABOLISM AND RELEASE OF DOPAMINE AND AMINO ACIDS FROM NERVE ENDINGS ISOLATED FROM SHEEP CORPUS STRIATUM

Abstract— Synaptosomes prepared from sheep corpus striatum showed a linear rate of respiration over a 90 min period of incubation in Krebs-bicarbonate medium containing glucose (10 mm ) and the rate of respiration was stimulated by electrical pulses. Dopamine was released from synaptosome beds to the medium by either electrical pulses or 56mm -K⁺ (10min), increasing 108% and 76% respectively above control levels of release. The presence of d- or 1-amphetamine (0.12mm ) in the incubation medium (40 min) increased the accumulation of dopamine in the medium by 310 and 275% respectively and 56mm -K⁺ also caused a significant increase in the release of glutamate, GABA and aspartate. Radioactively labelled dopamine was synthesized by the synaptosomes from l -[¹⁴C]tyrosine, l -DOPA or dl -DOPA, and electrical pulses caused a 35% increase in the rate of dopamine production from [U-¹⁴C] tyrosine. No increased release of [¹⁴C]dopamine in response to depolarizing stimuli was found to occur when synaptosome beds were transferred from medium containing radioactive precursors to fresh medium for further incubation (20 min). In the presence of 1- and d-amphetamine, accumulation of ¹⁴C-labelled doparnine in the incubation media was increased 129% and 380% respectively, the latter was partially depressed by absence of calcium from the medium. Three radioactively labelled metabolites formed by synaptosomes during incubation in dl -[2-¹⁴C]DOPA were detected; the major ones were dihydroxyphenylacetic acid and homovanillic acid and the third was unidentified. When the synaptosome beds were transferred to medium containing no radioactive precursors, it was found that labelled dihydroxyphenylacetic acid was 7 times more abundant than labelled dopamine in the incubation medium (20 min) and one-third as abundant in the synaptosomes. The dihydroxyphenylacetic acid n Ci/dopamine n Ci ratio was greatly affected by K⁺ stimulation, decreasing 52% and 34% in the incubation medium and synaptosomes respectively. A pathway of dihydroxyphenylacetic acid degradation was shown to occur through decarboxylation. These results are discussed in terms of the compartmentation of dopamine and its metabolism. It is proposed that one pool of dopamine is released by depolarizing agents and during the period of incubation it is replaced by synthesis from the endogenous tyrosine (19.5 nmol/100 mg protein) and not by the labelled dopamine in the synaptosome. The synaptosomal pool of dopamine which is radioactively labelled after pulse labelling with dl -[2-¹⁴C]DOPA appears to be prone to oxidation to DOPAC and homovanillic acid which are preferentially released from the synaptosomes.     

Effect of removing sialic acids from endothelium on the adherence of circulating platelets in arteries in vivo

The contribution of the net negative charge excess due to sialic acids on endothelium in preventing adhesion of circulating platelets in vivo was investigated in anaesthetized rabbits. Platelets in the rabbit's circulation were selectively labelled with radioactive 5-hydroxytryptamine in vivo. Segments of carotid arteries temporarily isolated from the circulation were perfused with one or other of two commercial preparations of neuraminidase; the opposite carotid artery was perfused similarly without the enzyme, as control. A neuraminidase preparation from Behringwerke free of proteolytic activity released sialic acid into the perfusate with a peak concentration after 10-15 min which decreased gradually later. A neuraminidase preparation from Sigma that contained demonstrable proteolytic activity released sialic acid similarly during the first hour and thereafter more sialic acid in a second peak. After blood flow through the carotids had been restored the adhesion of labelled platelets in the artery perfused with neuraminidase was compared with that in the artery perfused without the enzyme. The radioactivities were significantly higher in carotids that had been perfused with neuraminidase than in those that had been perfused without the enzyme. Neuraminidase perfusion had no effect on the production of prostacyclin by the carotids. Perfusion with acetylsalicylic acid before neuraminidase increased the adhesion of platelets significantly. It is concluded that diminution in electrostatic repulsion between circulating platelets and vascular endothelium from which the net negative charge excess due to sialic acids has been removed increases the adhesion of circulating platelets, irrespective of the production of prostacyclin by the arterial walls, and that inhibition of prostacyclin production augments this adhesion of platelets.     

Biosynthesis of nucleic acids in pea (Pisum sativum L.) buds released of dominance

Biosynthesis of nucleic acids in the pea buds released from apical dominance by decapitation has been studied. The rate of biosynthesis of all nucleic acid classes separated by means of methylated albuminkieselgur (MAK) column chromatography was found to be increased in released pea buds as judged by the extent of orotic-6-¹⁴C acid incorporation. This increase has been observed in all experimental points examined (15, 24, 36 and 48 h after decapitation), being the most pronounced 15 h after decapitation. After that a certain decrease of the specific radioactivities of nucleic acids has been registered, but even 48 h after decapitation specific activities of the main nucleic acid classes were higher in comparison with those found in dominant buds. Variations of the free pyrimidine nucleotide pools were about 14% suggesting that this parameter did not influence the extent of the uptake of labelled orotic acid.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polymo virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [³H]glycerol, [³H]myristate, [³H]choline or [³H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [³H]glycerol or [³H]myristate were stimulated by vasopressin, no significant decrease in the labelling of phosphatidylcholine was detected, but the labelling of phosphatidic acid was elevated. However, the labellings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [³H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells.Abbreviations BHK-21 cells baby hamster kidney-21 cells     

METABOLITES OF [3H]ACETATE BOUND TO SYNAPTIC VESICLES ISOLATED FROM RAT CEREBRAL CORTEX

Abstract— Synaptic vesicles were isolated from rat cerebral cortex after an intraventricular injection of [³H]acetate. The labelled substances bound to the synaptic vesicles were released by exposure to acid, separated from the vesicle membranes by Sephadex column chromatography and identified by thin-layer chromatography and thin-layer electrophoresis. The three major peaks of radioactivity were glutamate, glutamine and gamma-aminobutyric acid. Their presence in synaptic vesicles is consistent with the concept of an integration of energy metabolism, membrane regulation and synaptic transmission.     

Adhesion to porcine squamous epithelium of saccharide and protein moieties of Lactobacillus fermentum strain 104-S.

The mechanism by which Lactobacillus fermentum strain 104-S adheres to porcine squamous epithelium was investigated by studying the adsorption to epithelial cells, and control surfaces, of radioactively labelled material released from the bacterial cells by water extraction. The released material was fractionated by gel filtration and the adsorption of pronase-sensitive and -resistant material in the various fractions to porcine gastric tissue and the control surfaces of polystyrene and immobilized bovine serum albumin (BSA) was determined. The fraction with affinity for the epithelium was characterized by enzymic degradation, periodate oxidation, lipid extraction, and protein and carbohydrate analyses. The adsorption pattern of radioactively labelled crude released material mimicked the adhesion of whole labelled cells to polystyrene and to gastric squamous tissue pieces. On fractionation, the pattern of adsorption to polystyrene and BSA was different from that obtained for the tissue pieces. Considerably less labelled pronase-stable material bound to surfaces of polystyrene and BSA, as compared with the tissue, suggesting that the pronase-resistant component has a tissue-specific affinity. After pronase treatment of the fraction of M(r) about 20,000 (20 K) containing labelled components with affinity for the epithelium, only saccharides were detected. Radioactivity was lost after hydrolysis with HCl, and therefore this pronase-resistant labelled component must be a saccharide. It is concluded that protein moieties in the extract have an affinity for several surfaces, including polystyrene, and that saccharide moieties have a specific affinity for the gastric squamous epithelium.     

Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man

The nature of the metabolites excreted in the urine was investigated up to 48 h after oral and intravenous administration of 0.3 to 1.3 mg [1′,2′-³H₂]phylloquinone. The metabolites were water-soluble of which the major fraction consisted of glucuronide conjugates. A chromatographic comparison of the aglycone fragments released by β-glucuronidase and by dilute HCl revealed the presence of at least three labelled aglycones. The major aglycones obtained by enzyme hydrolysis consisted of at least two closely related organic acids which were not separated by adsorption thin-layer chromatography but one of which on treatment with dilute acid yielded a neutral metabolite with the chromatographic properties of phylloquinone γ-lactone. The results suggest that phylloquinone γ-lactone, the only previously isolated urinary metabolite of phylloquinone, is an artifact produced by the conditions of acid hydrolysis. Although the acid labile aglycone was the minor component of the two acid metabolites, its proportion in urine extracts as measured by conversion to the lactone, increased with the time after administration of labelled phylloquinone.     

Comparative effects of trypsin, collagenase and mechanical harvesting on cell membrane lipids studied in monolayer-cultured endothelial cells and a green monkey kidney cell line

Experiments are presented in which membrane lipids of endothelial cells in monolayer culture were labelled with [14C]linoleic acid. Approx. 90% of the radioactive label were incorporated into phospholipids. A comparison of various harvesting methods showed that during the disruption of the labelled endothelial cell monolayer, 0.25% trypsin and 0.125% trypsin (+0.01% EDTA) released 650 and 470% more radioactivity, respectively, than did 0.01% collagenase (+0.01% EDTA). Parallel studies were performed on a green monkey kidney cell line. In this case, 0.25% trypsin released 520% more radioactivity than did 0.1% collagenase (+0.01% EDTA), although 0.125% trypsin in the presence of EDTA (0.01%) was much less traumatic than trypsin alone, the released radioactivity being of the same order of magnitude as that for collagenase. Morphological studies on endothelial cell cultures failed to reveal any distinctive differences in surface morphology following the various enzyme treatments. The results suggest that collagenase treatment of endothelial cell monolayers is the least traumatic harvesting or subculturing method as far as the integrity of the lipids in the cell membrane is concerned.     

Degradation of indulin by Candida albicans.

Candida albicans utilized 14C (ring) labelled dehydropolymer of coniferyl alcohol, 14C-teakwood lignin and indulin and released p-hydroxybenzoic acid, vanillic acid, 3,4-dihydroxybenzoic acid and catechol as by products from lignin. Candida albicans produced catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, intra- and extracellular polyphenol oxidase and peroxidase during indulin degradation. The study suggests that Candida albicans degrades different types of lignin.     

The effect in vitro of ionizing irradiation and small rises in temperature on the uptake and release of labelled lipids by the human erythrocyte membrane

1. The effect of X-irradiation (50 000 rad) and an increase in temperature from 37 to 42 degrees C on the synthesis, uptake and release of labelled lipids by erythrocytes was studied in plasma incubations in vitro. 2. Both irradiation and a rise in temperature resulted in an inhanced synthesis of [32P]phosphatidic acid in the erythrocytes. 3. The uptake by the erythrocytes of 14C- and 3H-labelled cholesterol, [14C, 32P]phosphatidylethanolamine and [14C, 32P]phosphatidylcholine from plasma lipoproteins was increased by a rise in temperature but not by irradiation. These labelled lipids were apparently taken up in the ratio in which they were found in plasma. They were not released from the erythrocytes in the same manner.