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Thomas Jay Oord has recently proposed that God’s relationship to the world and its human inhabitants is well described as one of uncontrolling love, and that God is constitutionally incapable of intervening unilaterally in human lives. Consequently, prayer for God so to act is misjudged and inappropriate. This article argues that this conclusion runs counter to the biblical record, which sees God’s relationship with the world as sustaining and upholding, but also allowing for specific willed actions. However, God has given the world functional autonomy, which would be abrogated by interventions too predictable or too attributable. It is proposed that intervention by God is real and not uncommon, but often of concealed and unprovable provenance. God’s self-limitation with regard to intervention is seen as consistent but chosen, in contrast to Oord’s proposal of an ‘essential’ kenosis.

This article addresses the question of whether God intervenes in the regular workings of the physical world, either unprompted or in response to prayer. Its focus is on intervention that is not in the ordinary sense miraculous. It is proposed that, because humans undeniably have the ability to intervene and change the course of events, it is perverse and illogical to hold that the Creator cannot do likewise. But perhaps God chooses not to exercise his prerogative to intervene. Would not such intervention abrogate the God-given autonomy of the created world that makes its workings helpfully predictable and also accessible to scientific enquiry? Indeed, is it not unseemly for God to interfere with the day-to-day workings of the world? Jesus’ teaching about God’s nature suggests a nuanced approach, that of parenthood: one that includes the option to intervene, but is considerate and restrained. God’s intervention in response to prayer is the consistent premise of the Old and New Testaments, and it is probably unwise to put much store on human ability to discern what is and is not fitting for God to do. The validity and legitimacy of intercessory prayer are affirmed.

This paper draws attention to a substantial gap in the new Christian eschatology pioneered by Bishop Tom Wright and supported by many other theologians, some with solid scientific credentials. This eschatology sees a substantial degree of continuity between the present earth and a future earth-heaven to be inaugurated by the parousia of Jesus Christ. Lacking is a clear exploration of Christian expectations for the future of human and terrestrial history. This is a matter of urgent apologetic and pastoral concern.

The Five-Factor Model of Personality was empirically derived and is now ubiquitous in research and applied personality psychology. However it is presented in current textbooks as including an un-evidenced component: the judgement that, while the five traits are socially desirable qualities (except socially undesirable Neuroticism), low scorers are characterised by their deficiency in essential social skills, displaying unattractive undesirable personalities. This judgement is verbally reinforced by the value-laden designations of the five traits (excepting Extraversion). Thus the model ignores evidence for cognate but contrasting personality factors that are also desirable in individuals and for society. It is proposed that traits are better understood as coming in pairs, thus resembling allelic variant expressions of genes. Extraversion and Introversion would then each reference a quality with both positive and negative potential. The traits Openness, Agreeableness and Conscientiousness could become resp...

Self-knowledge is seen by many religious traditions as a key to growth in wisdom and maturity. It is also a key practical goal of all attempted taxonomies of the human personality. A distinctive feature of the MBTI® psychological type approach is its affirmation of the intrinsic value of both polarities of the key preferences, for example extraversion and introversion. The absence of value judgement in MBTI® psychological type is personally affirming, but could encourage complacency and narcissism. However, each type preference logically entails a weakness in using its polar opposite, and therefore preferences are also limitations, biases that cause and perpetuate blind spots and incapacities. Accepting this can lead individuals to recognise those situations in life where their type preferences are no guide or basis for behaviour and to work on their less preferred attitudes and functions. Thus self-knowledge can generate the self-control that biblical writers see as integral to wisdom and maturity.

The Myers-Briggs Type Indicator (MBTI®) is widely used as a staff-development tool in the business and voluntary sectors. Its Psychological Type approach is found to be a valuable aid to understanding self and others and thus to enhancing effective team-working. This continuing and growing popularity is surprising in view of the disdain with which MBTI® has long been regarded by the professional psychology community. The grounds of this hostility are here examined, revealing a remarkable convergence between the conceptual frameworks of MBTI® and its more orthodox counterparts in personality psychology, as well as several significant differences. The Type and Trait approaches both conclude that there are just four principal and independent components of the normal personality, and are in close agreement in identifying these four components. The differences are principally in the different theoretical frameworks of the Type and Trait approaches. An evaluation of these differences suggests that a harmonizati...

Myers–Briggs personality typing is widely used in the Christian church as an aid to individual self‐understanding and spiritual formation. However, some Christian leaders have expressed doubt about its validity in understanding human personality and also opposition to its use in nurturing spiritual growth. The aim of the work reported was to identify and examine the reasons for this negative stance towards personality typing. It was achieved by content analysis of published writings and, in a few cases, by correspondence with persons known to have a critical stance. The analysis showed five principal areas of concern: misuse of personality typing in spiritual formation; personality typing as a simplistic analysis; personality typing as a restrictive pigeon‐holing; unethical use of personality typing; and the Jungian derivation of personality type theory. Evaluation of this unease reveals significant semantic and epistemological issues and also a concern that personality typing, while not denying the tenet...

Drug delivery by means of polymer conjugates that are internalized into cells by endocytosis is now a viable therapeutic approach. Successful deployment of this model depends upon release of the free drug within the endosome/lysosome compartments and its efflux into the cytoplasm. The latter process involves the drug crossing the endosome/lysosome membrane, which is known to be impermeable to all large and many small molecules and which is equipped with numerous substrate-specific transporters that allow metabolites across. Passive diffusion is the only viable mechanism for most xenobiotics to cross the endosome/lysosome membrane. Studies are reported on the permeability of the rat liver lysosome membrane. These demonstrate that permeance of molecules correlates inversely with their hydrogen-bonding capacity, a function that can be calculated from inspection of structural formulae. It is deduced that drug molecules containing cationic and/or anionic functional groups, or numerous hydrogen-bonding moieties such as hydroxy, ether or carbonyl, will cross the lysosome membrane unacceptably slowly, but that many drugs will cross at a satisfactory rate. This conclusion is supported by the rather meagre data available on membrane permeability of lysosomes in situ within cells. Systematic experimental studies on the endosome membrane are lacking, but there is every reason to suppose that its permeability is similar to that of the lysosome membrane.

The membrane of the lysosome contains substrate-specific porters for a wide range of metabolites. Their physiological role is in promoting the efflux of the products of intralysosomal catabolism. With few exceptions, the specificity of these porters makes them unlikely candidates for the translocation of xenobiotics across the lysosome membrane. Where efflux from the lysosome is possible, it is likely to be accomplished by passive diffusion. Experimental studies on passive diffusion across the lysosome membrane have shown that its characteristics are similar to those of other biological membranes. Ease of permeation decreases with increasing hydrophilicity. Macromolecules and some highly hydrophilic molecules as small as sucrose are effectively non-permeant. The notional hydrogen-bonding capacity of molecules (an inverse correlate of oil:water partition coefficient) has been found a good predictor of permeance. Predictions of ease of permeation across lysosome membranes is of value when drug delivery strategies are contemplated that involve a drug-conjugate reaching the lysosome compartment and drug release there by the lysosomal enzymes. These strategies will be unsuccessful if the drug is unable to leave the lysosome and reach the cellular sites where its pharmacological action is required.

In order to study its fate in vivo, a low molecular-weight polylactide homopolymer was derivatized with a p-methoxyphenyl moiety, so as to make it susceptible to radiolabeling with 125 I . A low molecular weight polylactide–polyethyleneglycol copolymer capped with a p-methoxyphenyl residue was also synthesized. The derivatized polymers were successfully [ 125 I ]iodinated in organic medium. The radiolabeled products were freed from [ 125 I ]iodide by dialysis and shown to be stable for 24 h on incubation at 37°C in buffered saline or in blood. On longer incubation at 37°C in buffered saline the radiolabeled polylactide released [ 125 I ]iodide and [ 125 I ]iodinated 3-(p-methoxyphenyl)propionic acid. The radiolabeled copolymer was more stable on incubation at 37°C in buffered saline, but some [ 125 I ]iodide was released. The tissue distribution of radioactivity was determined 5 min, 1, 5 and 24 h after injecting male rats with 125 I -labeled homopolymer or copolymer. Intravenous, intraperitoneal and subcutaneous injection routes were employed. Further rats were injected with [ 125 I ]iodide, to aid interpretation of the data. After administration of labeled homopolymer, a high concentration of radioactivity was found in the liver tissue. The levels slowly decreased over 24 h, and the polymer was successively found in the small and large intestine and the faeces. This is probably indicative of excretion via the bile. Concurrently radioactivity was excreted in the urine. After administration of labeled copolymer, a high concentration of radioactivity was found in the liver and the residual soft tissue, the latter fraction containing two-thirds of the radioactivity one hour after injection. The precise tissue location that this result indicates was not identified. After 1 h radioactivity was excreted in the faeces, again probably via the bile, and in the urine. Tissue distributions after intraperitoneal or subcutaneous injections were concordant with the above results and interpretations, with the additional factor of slow clearance from the injection site.

The effect of supplementary L-methionine (Met) on the incorporation of methionine was evaluated in 9.5-day rat conceptuses cultured in vitro. Parallel experiments with L-leucine (Leu) were performed for comparison. Conceptuses were cultured for 24 hr in the presence of 3H-labeled Met or Leu, and the incorporation of radiolabel into the embryo and visceral yolk sac was measured. Supplementary Met proportionately increased the incorporation of Met, but supplementary Leu did not have as great an effect on the incorporation of Leu. A hypothesis is presented to explain these findings. It is proposed that Met, but not Leu, is a rate-limiting nutrient for organogenesis-stage rat embryos cultured in rat serum. The results are also discussed with reference to the established efficacy of supplementary folic acid in decreasing the incidence of neural tube defects in human populations and to claims that Met reverses certain teratogenic phenomena, both in vitro and in vivo.

It is known that intracellular proteolysis of exogenous protein in the visceral yolk sac (VYS) is a significant source of leucine for the fetus from days 8.5 through 17.5 post-conception in the rat. In contrast, little attention has been paid to the potential of the VYS to transport maternal free amino acids to the fetus. We investigated this question using the exteriorized 17.5-day rat fetus, enclosed within its amnion and VYS, but with its placenta attached to the uterus and still functional. We first demonstrated that [ 3 H] leucine in medium bathing the exteriorized fetus is transported by the VYS to the fetus and then exchanged with the leucine pool in the maternal circulation. This result indicates that the preparation remains viable for the duration of the experiment. We next injected a trace amount of [ 3 H] leucine intravenously into the 17.5-day pregnant rat and found that the concentration of radioactivity in the plasma of the exteriorized fetus was not much lower than that in plasma of non-exteriorized fetuses in the same litter (8–15 fetuses per litter). We interpret these results to indicate that the VYS transports free leucine to the fetus but that this process makes a minor contribution to the total transfer of free leucine into the 17.5-day rat fetus.

The permeability of rat liver lysosomes to some inorganic and aliphatic organic anions was investigated, using an osmotic-protection methodology. Lysosomes were incubated at 25 degreesC in 250 mOsm solutions of potassium salts of the anions, in the presence of valinomycin, and the latency of lysosomal hexosaminidase measured at intervals. Lysosomes suspended in 250 mM sucrose at 25 degreesC were stable for up to 4 h. When suspended in 250 mOsm solutions of potassium salts of inorganic acids, latency was lost at rates indicating anion permeance decreasing in the order thiocyanate, nitrate and iodidebromidechloridesulfate. This rank order does not correspond with the anion selectivity of any known anion transporter, and is closer to that of the lyotropic series. Results with the potassium salts of aliphatic organic acids indicate little correlation between permeation and hydrocarbon chain length, although formate was more rapidly permeant than acetate and its higher homologs. By contrast, oxalate was less permeable than other dicarboxylic acids. The presence of one or more hydroxy groups decreased permeance. A correlation between permeance and the acid's lowest pKa suggested that penetration was due principally to the entry of the undissociated acid, but there is evidence that the (much more abundant) singly charged anionic form is also significantly permeant.

In an attempt to explain the previous observation of the rise and subsequent fall in glycogen content of the rat visceral yolk sac during the latter half of gestation, the activities of glycogen phosphorylase, glucose-6-phosphatase and lysosomal alpha-glucosidase were measured. Glycogen phosphorylase was found to be present in the yolk sac and, as in adult rat liver, was predominantly in the 'a' (active) form. The specific activity of the enzyme was lower than in adult rat liver, when expressed per mg tissue protein or per mg tissue wet weight, but similar when expressed per mg tissue glycogen. Phosphorylase activity in yolk sac was similar at 16.5 and 18.5 days of gestation. Glucose-6-phosphatase activity was not detectable in the yolk sac at either 15.5 or 18.5 days of gestation. Two lysosomal enzymes, acid alpha-glucosidase and N-acetyl-beta-hexosaminidase, were shown to be present in the yolk sac at higher specific activity than in adult liver. Alpha-Glucosidase activity in yolk sac was similar at 15.5 and 18.5 days of gestation. It is concluded that the net degradation of yolk sac glycogen initiated around 18.5 days of gestation does not serve to provide glucose for the fetus, and may indicate an increased demand for metabolic energy within the yolk sac itself.

Summary Rat conceptuses on the 10th day of gestation were cultured for 27 h in whole rat serum. An addition of either [ 3 H]leucine or [ 3 H]leucine-labelled rat serum proteins was made once during the culture period, and the acid-soluble and acid-insoluble radioactivities of embryo and visceral yolk sac measured at harvesting. The extent of radiolabel incorporation into embryonic and yolk-sac proteins increased linearly with the duration of exposure of the conceptus to the radiolabelled leucine or radiolabelled serum proteins, indicating roughly constant rates of incorporation, per unit mass of tissue, throughout the culture period. The incorporation rates, expressed as clearances, were 0.73 and 0.78 μl/mg tissue protein/h for embryo and yolk sac, respectively, when the source was [ 3 H]leucine; and 1.8 and 1.3 μl/mg tissue protein/h, for embryo and yolk sac, respectively, when the source was [ 3 H]leucine-labelled serum proteins. It is estimated, from the known leucine and protein concentrations in serum, that protein contributed over 99 per cent of the leucine supplied to the conceptus for its protein synthesis. In parallel experiments, measurements were made on cultures conducted in the presence of an antiserum against rat visceral yolk sac (100 μg/ml). Antiserum profoundly inhibited incorporation of radioactivity into embryo and yolk-sac proteins, when the source was 3 H-labelled protein, a result consistent with the known ability of the antiserum to inhibit pinocytosis in the yolk sac. Antiserum also decreased incorporation from [ 3 H]leucine in the yolk sac, suggesting that a proportion of the free leucine entering the yolk sac does so by pinocytosis. The failure of antiserum to affect incorporation of [ 3 H]leucine into the embryo probably indicates that leucine can enter the embryo without the mediation of yolk-sac pinocytosis. The primacy of protein, as a source of amino acids for the organogenesis-stage embryo, is consistent with the serious effects, in terms of embryonic death and malformation, that result from the interruption of amino acid supply when either pinocytosis or lysosomal proteolysis in the yolk sac is inhibited.

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

The laxative action of phenolphthalein (5) is believed to result from induction of potassium and water efflux from the colon epithelium. In cultured cells, K+ efflux is promoted by 5 and by a contaminant (1) present in commercial phenol red. Six compounds with chemical structures related to those of 5 and 1 were tested for ability to induce the release of 86Rb from COS-7 cells preloaded with this isotope: 4,4'-(9-fluorenylidene)diphenol (2), 4, 4'-(9-fluorenylidene)dianiline, 4, 4'-(9-fluorenylidene)bisphenoxyethanol, 1,1'-bi-2-naphthol, 4, 4'-biphenol, and bis(4-hydroxyphenyl)methane. With one exception these compounds were all inactive at a concentration of 10 microM. However, 2 caused profound 86Rb efflux at concentrations as low as 100 nM. Concentrations of 5 1-2 orders of magnitude higher were needed to achieve similar levels of activity. The three compounds known to be active in this experimental system share a common feature that is absent in all the inactive compounds: a five-membered ring structure, one of whose carbon atoms is disubstituted with p-hydroxyphenyl residues. Because 2 and 5 are readily available, comparative studies on the mechanism of action of these biphenols at the cellular level can now be undertaken.

1. It has been proposed that phenolphthalein exerts its laxative effect via an intracellular cascade that begins with the activation of nitric oxide synthase (NOS) and ends with an inhibition of NaCl and water reabsorption from the colon. Phenolphthalein also promotes the release of potassium from cells, but it is not known how this is related to its effect on sodium and water uptake. 2. An established in vitro system was used to examine the role of nitric oxide (NO) in phenolphthalein-induced release of (86)Rb(+) from COS-7 cells. 3. Sodium nitroprusside, an NOS-independent NO source, was unable to mimic the effects of phenolphthalein and N(G)-nitro-L-arginine methyl ester, an NOS inhibitor, was unable to block the effect of phenolphthalein. 4. It is concluded that NO generation is not required for phenolphthalein-stimulated potassium release. It is proposed that the effect of phenolphthalein on cellular potassium release is mechanistically distinct from the effect on NaCl and water uptake by colonocytes.

Polyethyleneimine (PEI) is shown to destabilize isolated rat liver lysosomes, as indicated by a decrease in the latency of their acid N-acetyl-beta-glucosaminidase. PEI also inhibited the generation of radiolabeled digestion products from 125I-labeled bovine serum albumin endocytosed by rat visceral yolk sac in vitro. However, PEI did not greatly inhibit the endocytic uptake of a nondigestible fluid-phase substrate, fluorescein isothiocyanate (FITC)-dextran. It is hypothesized that PEI inhibits the adsorptive endocytosis of 125I-labeled bovine serum albumin, and thus its subsequent intralysosomal digestion, by competing with and displacing the labeled protein from its binding sites on the visceral yolk sac cell surface. This hypothesis suggests a plausible explanation for the ability of PEI to act as an efficient vector for gene and oligonucleotide transfer into mammalian cells. PEI present in the culture medium is carried into cells by adsorptive endocytosis. Concentrated thus on the endosome membrane, it permeabilizes this membrane and so affords DNA conjugated to the PEI an otherwise unavailable mode of access into the cytoplasm.

The visceral yolk sac (VYS) is responsible for a major part of the amino acid nutrition of the early post-implantation rat embryo and possibly also at the fetal stage of gestation. The mechanism involves endocytic uptake of proteins by the tissue's epithelial cells followed by intralysosomal digestion to amino acids. The amino acid so generated are used for protein synthesis in both the embryo and the VYS. Previous reports had indicated that the endocytic capacity of the VYS might be limited to exclude larger macromolecules. This study demonstrates that Percoll, which comprises 30-nm silica particles coated with polyvinylpyrrolidone (PVP), is as effectively captured by the 17.5-day rat VYS cultured in vitro as PVP itself. Uptake of 125I-labelled Percoll was progressive with time over 5 h and was inhibited by a low incubation temperature, 2,4-dinitrophenol (50 micrograms/ml), EGTA (5 mM), colchicine (10 micrograms/ml) or cytochalasin B (10 micrograms/ml). After uptake of 125I-labelled Percoll, VYSs released only 20 per cent of their radioactivity when re-incubated in fresh medium for 3 h. These data, and electron micrographs showing Percoll in intracellular vacuoles, are all consistent with uptake by endocytosis. Percoll's rate of uptake by the VYS indicates that, like 125I-labelled PVP, it enters the cell chiefly by fluid-phase pinocytosis. It is concluded that endocytosis by the VYS will efficiently capture even the largest globular proteins, and that previous indications of a relatively low size exclusion reflected the loosely coiled configuration of the synthetic polymers used in the earlier studies.

This book on lysosomes is appearing in 1996, 30 years after the publication of the landmark review by Christian de Duve and Robert Wattiaux in the Annual Review of Physiology. It so happened that I was visiting Dr. Wattiaux’s laboratory in Namur, Belgium, on the day the proofs of this review arrived in the mail. A vivid memory is his amusement that the key word in its title had been misspelled lusosomes.

The chapters in Section II of this volume review the catabolic potential of the lysosome, describing how the enzymes of the lysosome degrade the major classes of biopolymers. By a series of sequential hydrolytic steps, most macromolecules are digested to the monomer level. This chapter examines the fate of the end products generated in the lysosome and the role of the lysosome membrane in regulating metabolite flow from lysosome to cytoplasm.

Delivery Routes to the Lysosome. The Taxonomy of Lysosomes and Related Structures J.B. Lloyd. Origin of Lysosomal Proteins T. Braulke. Endocytosis E. Smythe. Autophagy E.G. Mortimore, et al. Selective Proteolysis: 70kD Heat Shock Protein and Ubiquitin-Dependent Mechanisms R.J. Mayer, F.J. Doherty. Metabolism in the Lysosome. Lysosomal Metabolism of Proteins R.W. Mason. Lysosomal Metabolism of Glycoconjugates B.G. Winchester. Lysosomal Metabolism of Lipids W.J. Johnson, et al. Lysosomal Nucleic Acid and Phosphate Metabolism, and Related Metabolic Reactions R.L. Pisoni. The Lysosome in Its Cytoplasmic Environment. Acidification of Lysosomes and Endosomes R.W. Van Dyke. Metabolite Efflux and Influx Across the Lysosome Membrane J.B. Lloyd. Lysosome Pharmacology and Toxicology R. Wattiaux, S. Wattiaux-De Coninck. Index.

The concept of the uncorkable liposome composed of phase-separated mixtures of a polymerized phospholipid and an enzymically digestible phospholipid has been investigated, using small unilamellar vesicles composed of mixtures of (polymerized) dienoylphosphatidylcholine (DENPC) and dimyristoylphosphatidylcholine (DMPC). Mixed liposomes, even those containing only 10% DENPC, were much more stable than DMPC liposomes, as indicated by the release of entrapped [3H]inulin or [14C]glucose. DMPC liposomes released entrapped solute on exposure to phospholipase A2, whereas mixed vesicles were resistant. The results are compared with those of an earlier study on monolayers of similar compositions. It is concluded that the liposomes, like the monolayers, are phase-mixed, and that uncorkable liposomes cannot be constructed from the phospholipid mixture employed. It is proposed that, until further experimental evidence is produced, the enzymatically uncorkable liposome must be regarded as a theoretical construct.

Methods are reported for the quantitative measurement of pinocytosis in human monocytes isolated from peripheral blood. The cells, in adherent culture in plastic wells, were exposed for periods of up to 48 h to culture medium containing 125I-labelled polyvinylpyrrolidone (50 micrograms/ml) and the pinocytosis enhancer suramin (500 micrograms/ml). Uptake of radiolabel was linear with time and was inhibited by colchicine (100 micrograms/ml), results that are consistent with uptake of radiolabelled substrate by pinocytosis but not with superficial adsorption of radiolabel. Similar results were obtained using a 125I-labelled vinylamine-vinyl-pyrrolidone copolymer as radiolabelled substrate. The rates of pinocytotic uptake of 125I-labelled polyvinylpyrrolidone (in the presence of suramin) and of 125I-labelled copolymer were measured at various stages of in vitro monocyte-to-macrophage maturation. In contrast to an earlier report, we found no consistent differences in pinocytotic activity between cells at different stages of differentiation.

Small neutral dipeptides such as Gly-Gly are known to cross the lysosome membrane rapidly. The mode of dipeptide translocation was studied, using an osmotic-protection method. Results with dipeptide analogues, such as ω-amino aliphatic acids and taurine, indicated that dipeptides do not cross the rat liver lysosome membrane by unassisted diffusion. Using seven pairs of dipeptide stereoisomers, the penetration of the l -isomer was always found to be much more rapid than that of the d -analogue. It is concluded that the lysosome membrane contains a porter that recognizes and transports l -dipeptides.

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

1.Previous studies have indicated that the permeability of polyethyleneglycol across the human intestine is anomalously high in comparison with the permeability of sugars with similar molecular mass. In consequence it has been proposed that two or more distinct mechanisms must exist for the translocation of these classes of molecule or, alternatively, that the molecular parameter determining rate of penetration is each molecule's minimum molecular dimension. 2.The notional hydrogen-bonding capacity of a molecule correlates well with oil–water partition coefficient and also, in a variety of experimental systems, with rate of passive diffusion across biological membranes. A molecule's hydrogen-bonding capacity is calculated by inspecting the structural formula and summing the individual theory-derived hydrogen-bonding capacities of the molecule's functional groups. 3.A classic set of intestinal permeability data that includes several ethyleneglycol oligomers and several sugars is re-analysed. A good correlation between permeability and hydrogen-bonding capacity is demonstrated. Specifically, there is no discontinuity between the polyethyleneglycols and the sugars. The data are compatible with a simple model in which all the molecules studied cross the intestine by passive diffusion across cellular membranes.

Mechanisms of Nutritionally Caused Congenital Malformations: The Role of Methionine in Counteracting Antiserum-Induced Embryopathy • 362

The cellular phenomena of pinocytosis and lysosomal digestion are described, with particular emphasis on the handling of soluble macromolecules by living cells. A drug delivery system based on poly(hydroxypropylmethacrylamide) conjugated to drug analogues by oligopeptide sequences has been evaluated. The conjugates, which are stable in the bloodstream, are captured by pinocvtosis and taken to the lysosomal compartment of cells. There theoligopeptide sequences are hydrolysed by the lysosomal enzymes and the drug analogues released. Targeting has been demonstrated by incorporation into the polymer of side-chains bearing moieties that lead to non-specific or cellspecific adsorptive pinocytosis.

Incorporation of 20% tyramine residues into its structure greatly increased the rate of pinocytosis of poly (α,β-(N-2- hydroxyethyl))- DL -aspartamide (PHEA) by rat visceral yolk sacs cultured in vitro. Both the parent macromolecule and the tyramine derivative (PHEA-tyramine) were captured by adsorptive pinocytosis, the higher affinity of the derivative for the yolk sac plasma membrane being responsible for its greater rate of capture. Using 125I-labelled PHEA-tyramine, the relationship between substrate concentration and rate of capture was determined, it was also shown that following internalization, the PHEA-tyramine linkage is resistant to intracellular hydrolysis. Fluorescence micrographs were consistent with capture of both substrates being by pinocytosis and illustrated the highly efficient concentration of the tyramine derivative by yolk sac endodermal cells.

Summary A series of twenty two N-(2-hydroxypropyl) methacrylamide copolymers, each containing a different, potentially degradable side chain, were incubated with rat liver Tritosomes. Four of the side chains were digestible as judged by the liberation of a terminal 4-nitroaniline residue. The pH optimum for the degradation of the side chain -e-aminocaproyl-phenylalanyl-4-nitroanilide was in the range 5.75–6.5 over the first hour of incubation and somewhat lower (pH 5.5–6.0) after this time. The degradation of the above side chain had a Km value of 58.3 mg/ml.

N-(2-Hydroxypropyl)methacrylamide copolymers are considered to be a potential drug delivery system. To fulfil this role the drug-polymer linkage must be susceptible to intralysosomal hydrolysis. Taking p-nitroanilide as a drug analogue, copolymers were synthesized bearing oligopeptidyl-p-nitroanilide side-chains designed to match known specificities of the lysosomal enzymes cathepsin L or cathepsin D. Degradation of side-chains by rat liver lysosomal enzymes (measured by monitoring terminal p-nitroaniline release) occurred only in the presence of reduced glutathione (5 mmol/l) and was effectively inhibited by leupeptin, indicating the involvement of thiol-proteinases in every case. Depending on side-chain composition, between 20 and more than 50% of the terminal p-nitroaniline residues were liberated during a 5 h incubation. It has also been shown that 1) a polymer molecule may contain side-chains of a single type that are nevertheless differentially susceptible to lysosomal hydrolysis; 2) two of the side-chains studied liberate only a p-nitroaniline residue, whereas the others also release amino-acyl-p-nitroanilides; 3) the cleavage of all side-chains displays a broad pH optimum pH 5 to pH 7; 4) the Michaelis-Menten constant Km for side-chain cleavage varied between 26,1 and 143,2 mg/ml, depending on the amino acid sequence of the side-chain.

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.

Poly- l -lysine, poly- l -α-ornithine, poly- l -glutamic acid, dextran, DEAE-dextran and dextran sulphate all fail to affect greatly the rate of pinocytic uptake of 125 I-labelled polyvinylpyrrolidone by 17.5-day rat visceral yolk sac or rat peritoneal macrophages cultured in vitro. It is concluded that these agents do not much affect the rate of pinocytic ingestion of liquid. Polycations accelerate the accumulation of colloidal 198 Au in both systems, and this is ascribed to the formation of substrate · modifier complexes which either adsorb to plasma membrane, and thus gain rapid entry, or initiate another mode of endocytosis. Pinocytic uptake of formaldehyde-denatured 125 I-labelled bovine serum albumin was accelerated by poly- l -lysine and poly- l -ga-ornithine in the macrophage, buth inhibited in the yolk sac.

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.

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 [125I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 ug/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 polyvitiylpyrrolidone. 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 teratogenests might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.

The membrane around the lysosome is more than a cordon sanitaire , protecting the cytoplasm from the dangerous brew of hydrolytic enzymes in these organelles. Its permeability properties are tailor-made to enhance the efficiency of lysosome function. Specific transporters exist in the membrane for certain metabolites.

DBA2 mice were inoculated i.p. with 10(5)L1210 cells. Animals subsequently treated with daunomycin (single i.p. dose, 0.25-5.0 mg kg-1) all died. The maximum increase in mean survival time observed was approximately 135%. Animals treated with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to daunomycin (DNM) showed a significant increase in mean survival time when the polymer-drug linkage was biodegradable (i.e., Gly-Phe-Leu-Gly). Such treatment also produced a number of long term survivors (greater than 50 days). In contrast, HPMA copolymer conjugated to DNM via a non-degradable linkage (Gly-Gly) produced no increase in survival time relative to untreated control animals. The effect observed with biodegradable HPMA copolymer-DNM conjugates was dependent on the concentration of conjugated drug administered (optimum greater than 5 mg kg-1); the frequency of administration (multiple doses were more effective than single); the timing of administration (single doses given on days 1 and 3 were most effective); and the site of tumour inoculation and route of drug administration. Biodegradable HPMA copolymer-DNM conjugates administered i.p. were active against L1210 inoculated s.c. at higher doses than required to curb a peritoneal tumour. Under certain experimental conditions polymer-DNM conjugates containing fucosylamine or galactosamine proved more active than conjugates without the carbohydrate moeity. The mechanism of drug-conjugate action in vivo is at present unclear. Radioiodination of polymer showed approximately 75% of polymer-drug conjugate to be excreted 24 h after i.p. administration. Synthesis of HPMA conjugates containing [3H]DNM showed that polymer containing Gly-Gly-[3H]DNM was excreted (60% of radioactivity in the urine, 24 h) in macromolecular form. In contrast polymer containing Gly-Phe-Leu-Gly-[3H]DNM was largely excreted in the form of low molecular weight species.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

A block copolymer (6) with both hydrophilic and hydrophobic regions was synthesized, in order to examine its interaction with model membranes and its uptake by living cells. The copolymer comprised poly(ethylene oxide) and poly(L-lysine) with 50 mol-% substitution of the e-amino groups with palmitoyl groups. To permit 125I-labelling, p-methoxyphenyl residues (1–4 mol-%) were incorporated into the block copolymer and into a poly(ethylene oxide) used for comparison. Sudan Red 7 B solubilization studies indicated that the block copolymer, but not the homopolymer, forms micelles. Differential scanning calorimetry of dipalmitoyl-phosphatidylcholine liposomes indicated that the block copolymer interacts with and probably penetrates lipid membranes. Both poly(ethylene oxide) and the block copolymer were captured by rat peritoneal macrophages in vitro, and inhibitor studies indicated that uptake of both polymers was by pinocytosis. Rates of uptake were indicative of adsorptive pinocytosis, and it is concluded that both poly(ethylene oxide) and the block copolymer present a largely hydrophilic aspect which interacts positively with the cell surface.

Soluble synthetic polymers have potential as targetable carriers of pharmacological agents. Here we report that incorporation into poly[N-(2-hydroxypropyl)methacrylamide)] of an oligopeptide side-chain terminating in galactose enhanced the polymer's pinocytic uptake from the rat bloodstream by the liver. Within the liver lysosomes enzymic digestion led to the intracellular release of a drug analogue also bound to oligopeptide side-chains of the polymer.

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.

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.

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of 125I-labelled 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.

Preparation and fractionation of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers containing oligopeptide side-chains crosslinked with dityrosylhexamethylenediamine to a level below the gel point has been described previously [1]. Fractions of mean molecular weight ( M w ) from 34,000 to > 400,000 were radiolabelled with 125I and used to study the effect of molecular weight on the fate of polymer in vivo. The blood clearance and body distribution (60 min) of each molecular weight fraction was measured after intravenous administration to rats. Blood clearance was clearly related to molecular weight. In addition the body distribution of unfractionated HPMA copolymer ( M w = 190,000) was monitored at various times after intraperitoneal, subcutaneous or oral administration to rats. Copolymer administered intraperitoneally progressively moved out of the peritoneal cavity into the bloodstream, and after 24 h most radioactivity was recovered in the urine. Sepharose 4B/6B column chromatography of urine samples showed that most radioactivity represented low molecular weight polymer but low molecular weight degradation products were also present. After subcutaneous administration polymer persisted at the injection site, gradually moving away over 24 h. Material entering the body was recovered principally from the bloodstream, eventually being excreted in the urine and faeces. Oral administration led to rapid transfer of radioactivity along the gastrointestinal tract, after 24 h most radioactivity being recovered in the urine and faeces. Sepharose 4B/6B chromatography of radioactivity recovered from the caecum showed that extensive degradation of copolymer had taken place. Incubation of 126I-labelled HPMA copolymers in rat plasma and a mixture of rat liver lysosomal enzymes, followed by analysis with Sepharose 4B/6B or Sephadex G-15 chromatography, showed that the copolymers used in this study were relatively stable in plasma but degradable by lysosomal enzymes.