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The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of 125I-labeled PVP and colloidal 198Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [14C]DIVEMA and 125I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.

Hickman & Neufeld [(1972) Biochem, Biophys. Res. Commun. 49, 992-999] have proposed that lysosomal enzymes reach the lysosomes by means of exocytosis and subsequent pinocytic reincorporation. The results leading to this conclusion are re-assessed and an alternative explanation is advanced that relates to the necessity for membrane recycling in endocytic cells.

Uptake of L- and D-alanine by the rat visceral yolk sac has been studied in vitro in incubations of short duration. It is concluded that much of the uptake of D-alanine is due to fluid-phase pinocytosis and that the plasma membrane L-alanine porter is more stereospecific than has hitherto been supposed.

Low temperature,2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of 125I-labelled polyvinylpyrrolidone, 125I-labelled bovine serum albumin or colloidal 198 Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the presence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.

Pinocytosis is a widespread phenomenon and involves the internalization of large amounts of plasma membrane. Much of this membrane is probably returned to the plasma membrane in vesicular form, and reasons are advanced why this recycling may occur chiefly before pinosome--lysosome fusion and may be related to vacuole fusion events. Some internalized membrane must nevertheless enter the lysosomal compartment and, in order to maintain a steady state, must be removed at the same rates as it enters. This constant lysosomal involution probably occurs by the budding of vesicles either inward (for digestion) or outward (for return to the plasma membrane). The former process allows for the transfer of cytosol components into the lysosome. Quantitative studies on pinocytosis have shown that selective substrate capture is commonly achieved by the adsorption of substances to the plasma membrane being internalized. In contrast the basal rate of uptake of liquid and membrane is not easily modified. If similar considerations apply to uptake by the quasi-pinocytic lysosomal involution, modification of proteins leading to adsorption to the cytoplasmic face of the lysosome could be the rate-determining step in the degradation of endogenous cytoplasmic proteins by lysosomes.

Low temperature, NaF and 2,4-dinitrophenol could each abolish the pinocytic uptake of 125I-labelled poly(vinylpyrrolidone) or colloidal [198Au]gold by rat peritoneal macrophages cultured in vitro. Cytochalasin B caused only partial inhibition, even at 10 microgram/ml, and colchicine (10 or 25 microgram/ml) inhibited uptake of colloidal [198Au]gold much more than that of 125I-labelled poly(vinylpyrrolidone). Dibutyryl cyclic AMP and ouabain were without effect on uptake of 125I-labelled poly(vinylpyrrolidone), and slight stimulation was seen with ATP and theophylline. Uptake of 125I-labelled poly(vinylpyrrolidone) was abolished by EGTA (5mM), but restored by adding CaCl2 (5mM). The results appear not to support the conventional criteria for the division of pinocytic phenomena into macropinocytosis, requiring a metabolic energy supply and cytoskeletal components, and micropinocytosis, requiring neither.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopecek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518-521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0-11.6 mol%) were injected intravenously into rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80-90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosinamide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37 degrees C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.

The rat visceral yolk sac is shown to possess a sodium-independent, phloretin-sensitive, and phlorizin- and ouabain-insensitive transport system for hexoses. The rate of uptake of (3H)2-deoxy-D-glucose was measured in vitro and shown to be greatest on the 12th day, decreasing progressively with increasing gestational age up to the 20th day. Little uptake of 3-O-methyl-D-glucose, alpha-methylglucoside or L-glucose occurred. On uptake by the visceral yolk sac, 2-deoxy-D-glucose was phosphorylated, leading to considerable accumulation of this sugar. Several sugars inhibited 2-deoxy-D-glucose uptake as follows: D-glucose = mannose greater than fructose greater than galactose greater than xylose greater than fucose.

Trypan blue at 50 microgram/ml stimulates the pinocytic uptake of 125I-labelled PVP, but not of colloidal 198Au or formaldehyde-denatured 125I-labelled bovine serum albumin, by the 17.5-day rat visceral yolk sac incubated in vitro. Neither Trypan blue nor a combination of the dye with 125I-labelled PVP stimulated the rate of pinocytosis of liquid by the tissue. Trypan blue itself was shown to enter the yolk-sac cells by adsorptive pinocytosis. It is proposed that an interaction between Trypan blue and 125I-labelled PVP enables the latter substrate to enter the cells adsorptively by so-called 'piggy-back' pinocytosis.

Synthetic 125I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [125I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.

[3H]Leucine-labelled haemoglobin was prepared from rat reticulocytes incubated in the presence of [3H]leucine. Conceptuses from 9-5-day pregnant rats were incubated in vitro tox 48 h, with [3H]leucine-labelled haemoglobin present for the final 12, 8, 4, 2 or 0.5 hours. Radioactivity accumulated in visceral yolk sac and in embryonic tissue. When exposure to labelled haemoglobin was for only a short period before harvesting, all the radioactivity found in the embryo and most of that found in the visceral yolk sac trichloroacetic acid-soluble (i.e. associated with free amino acid rather than with protein). After longer exposures the proportion of radioactivity that was acid-soluble decreased to minimum values of about 20%. SDS-polyacrylamide gel electrophoresis of the protein-associated radioactivity in visceral yolk sac and embryo was performed. After exposure to labelled haemoglobin for 1 h only prior to harvesting, the yolk sac contained a single peak of radioactivity coincident in mobility with haemoglobin. The embryo contained no protein-associated radioactivity. After exposure to labelled haemoglobin for 12h, many protein bands in both yolk sac and embryo were radiolabelled. Thus a single radiolabelled protein pinocytically captured by the visceral yolk sac can give rise to the presence of many labelled proteins in embryo and visceral yolk sac. These results indicate that the source protein underwent proteolytic digestion and that the amino acids generated were re-utilized for protein synthesis in both embryonic and visceral yolk-sac cells.

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.

Pinocytic uptake of 125I-labeled bovine serum albumin by 17.5-day rat visceral yolk sac cultured in vitro has been examined. Uptake was followed by intracellular digestion and, after an initial period, the content of radioactivity in the tissue itself remained constant during the incubation. Radiolabel was returned to the culture medium predominantly as (125I)iodotyrosine; exocytosis of undigested protein did not occur. The rate of uptake of labeled protein, which was constant within an experiment and reproducible between experiments, was much higher than that of a nondigestible macromolecule, 125I-labeled polyvinylpyrrolidone. The higher rate of uptake was a consequence of the protein entering the cells chiefly by adsorption to the plasma membrane being internalized; 125I-labeled albumin did not stimualte, nor did 125I-labeled polyvinylpyrrolidone inhibit pinocytosis. Different preparations of 125I-labeled albumin had characteristically different rates of uptake, probably reflecting differences in affinity for plasma membrane receptors. The physiological significance of the findings is discussed.

Synthetic polycations cause a stimulation in the rate of tissue accumulation of colloidal 198Au by the rat visceral yolk sac (at 17.5 days of gestation) and rat peritoneal macrophages cultured in vitro. The mechanism of stimulation has been elucidated in these two cell types by using a dual-substrate technique, and by examining the differential effects of poly(D-lysine) and poly(L-lysine) and of metabolic and cytoskeletal inhibitors. Polycations cause aggregation of colloidal 198Au in the culture medium and increase its affinity for the plasma membrane. In the rat peritoneal macrophage this polycation-colloidal gold complex is pinocytosed, thus enhancing the intracellular accumulation of the radio-labelled substrate. In contrast, the rat visceral yolk sac cannot internalize this complex, and so the substrate accumulates extracellularly. This mechanism of polycation modification affords the opportunity for differential uptake of a substrate into distinct cell types.

Benzodiazepines in the blood samples typical of forensic science work are recovered from 100-250 microliters amounts of blood (diluted with aqueous sodium octyl sulphate to suppress protein binding) onto microcolumns of Porapak-T, and finally eluted into 60-microliters volumes of aqueous acetonitrile. The eluates may be taken directly for analysis by high-performance liquid chromatography (HPLC) with reductive amperometric detection at a pendent mercury drop electrode held at potentials down to -1.2 V vs. Ag/AgCl. For high sensitivity work the electrode is preceded by a coulometric detector fitted with porous carbon electrodes held at 0 V (proprietary reference electrode). The technique detects all of the commonly encountered benzodiazepines and others except clobazam, which contains no azomethine group. The detection limits generally are in the range 1-5 ng/ml (40-200 pg HPLC-injected) in hemolyzed human blood, with recovery values of 84-95%, depending on the actual benzodiazepine, over the range examined (less than or equal to 2.14 micrograms/ml). The respective values for the metabolites of nitrazepam are 8-12 ng/ml and 75-84%. The technique is very much less susceptible to the interferences afflicting other commonly applied techniques, and facilitates considerably the analysis of degraded samples.

Contrary to previous reports, commercially available 1000-nm latex beads were found to be labelable with 125I, yielding a product that retained its radiolabel on storage at 4 degrees C and when incubated in tissue-culture media. This finding permitted a radiochemical method to measure phagocytic uptake of latex particles by rat peritoneal macrophages cultured in vitro, and a direct comparison with the established method of particle counting by light microscopy. The two methods yielded closely similar data, demonstrating that the (much more convenient) radiochemical method for quantitating phagocytic uptake is both feasible and reliable. The kinetics of phagocytic uptake of the latex particles and the effect of low temperature and metabolic inhibitors (sodium fluoride and 2,4-dinitrophenol) are described. Ongoing phagocytosis did not alter the rate of fluid-phase pinocytosis by macrophages.

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.

In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porter's ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct.

A method is described for the in vitro culture of 17.5-day rat visceral yolk sac. Tissue survival was good as judged by light and electron microscopy. The rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone by the tissue was constant both within and between experiments. Within the concentration range 0.15-24 mug/ml, the 125I-labeled polyvinylpyrrolidone neither stimulated nor inhibited pinocytosis. The system offers many advantages in the quantitative study of the physical basis of pinocytosis.

The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.

Improved density-gradient methods, using Percoll or Nycodenz, have recently been introduced for the isolation of human monocytes, but the capacity of cells thus isolated to differentiate into macrophages has not been systematically studied. We have compared Percoll and Nycodenz methods for the isolation of monocytes from human blood. The Nycodenz method yielded a monocyte population of high purity, but the yield was low. The Percoll method gave almost quantitative yield of monocytes, and the contaminating cells, mostly lymphocytes, were readily washed away after allowing the monocytes to adhere to a plastic surface. The Percoll method was then successfully scaled up, providing a simple method to obtain the monocytes from 180 ml blood. These monocytes were maintained in culture and their capacity to mature into macrophages was studied, using the following criteria: increase in cell size and protein content, increase in specific activity of hexosaminidase, differential hexosaminidase release on exposure to opsonized zymosan and unopsonized polystyrene beads, loss of peroxidase activity, and development of fluoride-insensitivity by the cells' cytochemically demonstrable esterase. The cells also displayed morphological changes typical of the monocyte-to-macrophage transition. The procedures reported constitute a simple and reliable method for the production of human macrophages in increased yield.

Previously it has been shown (Duncan, R., Starling, D., Rypácek, F., Drobník, J. and Lloyd, J.B. (1982) Biochim. Biophys. Acta 717, 248-254) that incorporation of tyramine residues into poly (alpha, beta-(N-2-hydroxyethyl]-DL-aspartamide (PHEA) greatly increases its rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro. Here we describe the relationship between the tyramine content (1.2-21.9 mol%) of modified PHEA and its rate of uptake by yolk sacs. Above a level of substitution of approximately 10 mol% the rate of uptake rises rapidly, and the concentration-dependence of capture is indicative of uptake by adsorptive pinocytosis. Serum proteins were shown to compete effectively for membrane binding sites, indicating a nonspecific interaction of PHEA-derivatives with the yolk sac membrane. PHEA derivatives of the same tyramine content, but of different mean molecular weights (Mr), were captured at the same rates.

Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in rho-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of rho-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of rho-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Gly-Phe-NAp where 5.1% of the bound rho-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.

Conceptuses from 9.5-day pregnant rats were cultured for 48 h in heat-inactivated homologous serum to which leupeptin, a specific inhibitor of the lysosomal cysteine-proteinases, was added for the final or the penultimate 6 h. The presence of leupeptin (25 micrograms/ml or above) increased the protein content of yolk sacs at harvesting to approximately twice the control value. The protein content of the embryo at harvesting was lower than that of controls. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. The presence of leupeptin did not affect the rate of uptake of the radiolabelled macromolecule by the yolk sac, nor facilitate its entry into the embryo. When formaldehyde-denatured 125I-labelled bovine serum albumin was added to the culture medium for the final 6 h of culture, little radioactivity was found in the yolk sac at harvesting, and barely any was found in the embryo. Trichloroacetic acid-soluble radioactivity was found in the culture serum. The presence of leupeptin sharply increased the levels of radioactivity in the yolk sac (but not the embryo) and sharply decreased the acid-soluble radioactivity of the culture medium. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in both yolk sac and embryo at harvesting. The presence of leupeptin increased the amount found in the yolk sac and decreased that found in the embryo. These results are interpreted as follows. Leupeptin enters the lysosomes of the yolk sac, inhibiting their cysteine proteinases. The digestion of proteins pinocytosed by the yolk sac is consequently inhibited, resulting in the accumulation of protein by the yolk sac and a decreased flow of amino acids to the embryo. Leupeptin (50 mg/kg), injected into pregnant rats at either 8.5 days or 9.5 days of gestation, induced congenital malformation in the offspring. It is proposed that leupeptin exerts its teratogenic action by inhibiting proteolysis in the lysosomes of the yolk sac, and so depriving the developing embryo of its supply of amino acids at a critical stage of development.

The glycogen content of the rat visceral yolk sac was determined between 13.5 and 20.5 days of gestation by the best available colorimetric method. The concentration of glycogen in the tissue increased ten-fold between 13.5 and 18.5 days, to reach a value similar to that for mammalian muscle, but then decreased by 50 per cent between 18.5 and 20.5 days. Determination of the iodine-iodide spectra and fractionation of the glycogen particles by a novel sodium citrate centrifugation method indicated broad similarities between the structures of glycogen particles, isolated by a mild phenol-water method, from the yolk sac and the liver of the rat. However, the proportion of 'high'-molecular-weight glycogen in the yolk sac increases between 18.5 and 20.5 days, as a result of the preferential loss of 'low'-molecular-weight glycogen, so that at term the proportion approaches that found in liver glycogen.

Cystinotic fibroblasts transferred from 37 degrees C to 28 degrees C accumulated additional cystine over the period from 4 to 7 days of incubation at 28 degrees C, after which the additional cystine was lost; warming (to 37 degrees C) of cells with elevated cystine stores led to rapid cystine loss. These results, taken together with previously published data showing cystine release from cystinotic fibroblasts incubated at above-normal temperature, are interpreted as indicating the presence in the cystinotic fibroblast lysosome membrane of a cystine-porter whose efficacy is increased by an increase in membrane fluidity. This porter may be the residual activity of the cystine porter that is known to be deficient in cystinosis, or it may be a second as yet unrecognized porter. It is further proposed that this porter is responsible for the presumed efflux of cystine from cystinotic lysosomes.

Thirty clones producing monoclonal antibodies (MCAs) to rat visceral yolk sac (VYS) antigens have been prepared. These MCAs localized by immunofluorescence in the VYS endoderm in vitro and were tested for developmental toxicity by intraperitioneal injection of ascites fluid into pregnant rats on day 9 of gestation. Five of the hybridomas produced MCAs that induced embryonic death, malformation, and growth retardation; the other MCAs had no developmental toxicity. Five MCAs, three teratogenic and two nonteratogenic, were tested for their ability to inhibit pinocytosis in the isolated day 17-VYS. Only the teratogenic MCAs were inhibitory, providing further evidence for the hypothesis that teratogenic antibodies interfere with the nutritional supply to the embryo.

Optimal assay conditions are described for plasma alpha-galactosidase, beta-glactosidase, beta-glucuronidase, alpha-mannosidase, alpha-glucosidase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, N-acetyl-alpha-glucosaminidase, acid phosphatase and arylsulphatase A. The levels of these activities in normal adults and children, and the stabilities of the activities on storage at -20 degrees C or 4 degrees C, are reported. The levels of these enzymic activities in plasma from patients with Fabry, Pompe, Sanfilippo A, Sanfilippo B, Tay Sachs and Hunter diseases, GM1-gangliosidosis and metachromatic leucodystrophy are described, and the possibility of using plasma hydrolase activities in the diagnosis of these conditions is discussed.

Rat peritoneal macrophages in vitro capture 125I-labelled polyvinylpyrrolidone entrapped within either negatively or positively charged liposomes more rapidly than they do the free macromolecule. The uptake of negatively charged liposomes was linear with time over 10 h, whilst the uptake of positively charged ones, although more rapid, was more transient. Neither type of liposome was taken up in the presence of 2,4-dinitrophenol (100 microgram/ml), and 5 mM calcium chloride increased the uptake of negatively charged liposomes. The enhanced uptake of 125I-labelled polyvinylpyrrolidone when presented in liposomes must have been a consequence of entrapment rather than of a simple interaction between lipid and polyvinylpyrrolidone since the presence of the lipids employed or of empty liposomes had no effect on the uptake of unentrapped 125I-labelled polyvinylpyrrolidone.

An increase in the concentration of serum resulted in a very marked increase in the rate of fluid-phase pinocytosis in rat peritoneal macrophages, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. In contrast, the rate of uptake of colloidal [198Au]gold, an adsorptive substrate, decreased when the serum concentration was increased. This lessened uptake of colloidal gold must be due to competition by serum components. Studies on the specificity of pinocytosis of simple proteins, using 125I-labelled H4 and M4 forms of porcine lactate dehydrogenase, and formaldehyde-denatured and untreated bovine serum albumin as substrates, suggest that positive charge and hydrophobicity determine adsorptive pinocytosis of simple proteins in peritoneal macrophages. The rate of fluid-phase pinocytosis and the specificity of adsorptive pinocytosis in peritoneal macrophages are very similar to those reported for Kupffer cells.

Suramin (250 mg/kg) and sodium aurothiomalate (100 mg/kg) both induced congenital malformations in the offspring following treatment of pregnant rats at either 8.5 or 9.5 days of gestation. Conceptuses from 9.5-day pregnant rats were cultured for 48 h in homologous serum to which either suramin or sodium aurothiomalate was added for the final 6 h. The presence of suramin up to 5 mg/ml had no effect on the protein content of yolk sacs at harvesting, but at 10 mg/ml caused a significant decrease. In contrast sodium aurothiomalate increased the protein content of yolk sacs at harvesting, in a concentration-dependent manner up to 100 micrograms/ml. Neither suramin nor sodium aurothiomalate significantly affected embryo protein content. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. When suramin (2-10 mg/ml) was present for the final 6 h of culture, the quantity of radioactivity measured in the yolk sac at harvesting was significantly decreased in a concentration-dependent manner. No radioactivity was detected in the embryos. Sodium aurothiomalate had no effect on the uptake of 125I-labelled polyvinylpyrrolidone. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in the conceptus (both yolk sac and embryo) at harvesting. Suramin (5 mg/ml), present for the final or penultimate 6 h, significantly decreased the uptake of radioactivity into conceptuses and caused a significant increase in the proportion of the captured radiolabel that was associated with the yolk sac. Sodium aurothiomalate (25 or 500 micrograms/ml) had no effect on the total uptake of radio-label but caused a significant increase in the proportion of total radioactivity captured that was associated with the yolk sac. These data indicate that suramin, by interfering with both the uptake and intralysosomal digestion of protein, and sodium aurothiomalate, by inhibiting digestion of captured protein, disturb the normal pathway of yolk sac-mediated protein utilization with a consequent diminution of the supply of amino acids to the conceptus. The effects of suramin are seen only at high concentration, those of sodium aurothiomalate at much lower concentrations. It is likely that the two drugs exert their teratogenic action by their effects on the yolk sac nutritional pathway with resultant amino acid deprivation of the conceptus at a critical stage of development.

Recently described microcolumn cleanup and high-performance liquid chromatography with electrochemical reductive mode detection have facilitated the determination of benzodiazepines in contaminated and degraded blood samples. Examples from an extensive application of the procedure to British forensic science casework are given here.