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Biomedical subjects

M K Pratten

Publications and source records attributed to M K Pratten.

At least 37 records · Page 2Linked to original sources
PubMed1995

Translactational induction of CYP4A expression in 10.5-day neonatal rats by the hypolipidemic drug clofibrate.

Lactating mothers of 7.5-day neonatal rats were injected intraperitoneally with 500 mg kg-1 clofibrate for 3 consecutive days at 24-hour intervals; 24 hours after the final injection, the maternal cytochrome P450 4A (CYP4A) mRNA levels had risen 14- and 2.5-fold above the constitutive levels of expression seen in the liver and kidney, respectively. Lactational transfer of clofibrate to the suckling 10.5-day litter was demonstrated by the 15- and 5-fold elevation observed in the neonatal hepatic and renal CYP4A mRNAs, respectively, following suckling from drug-induced mothers. A significant decrease in the relative liver weights of these neonatal pups was seen following clofibrate exposure via maternal milk, in total contrast to the normally observed increase in liver/body weight ratios of rats treated with clofibrate. Western blot analysis using a polyclonal goat anti-rat CYP4A1 antibody also demonstrated a rise in the CYP4A protein levels in both the mothers and their litters following maternal clofibrate treatment.

Animals↗
PubMed1992

Insulin and insulinlike growth factors in embryonic development. Effects of a biologically inert insulin (guinea pig) on rat embryonic growth and development in vitro.

Congenital anomalies occur up to four times more frequently in diabetic pregnancy than in the nondiabetic population. Although past work has shown that maternal hyperglycemia and hyperketonemia may increase embryonic abnormalities, recent experimental evidence suggests that low insulin levels may also contribute to diabetic embryopathy. This study investigated the effects of guinea pig serum (whose insulin is inactive in rat systems) on rat embryonic growth and development in culture. Supplementation of guinea pig serum with pork insulin at low (1 ng/ml) and high (5 ng/ml) physiological concentrations and insulinlike growth factors (IGF) I and II were also studied. Culture of rat embryos from the early headfold stage in guinea pig serum resulted in poor embryonic growth and development with a 92% rate of anomalies. Supplementation of guinea pig serum with zinc-binding pork insulin significantly improved rat embryonic growth and development (46% anomaly rate) especially between the first 5 and 21 h of the period of organogenesis. This evidence supports our most recent findings that low insulin levels, as encountered in untreated diabetic pregnancy, may contribute to the increased risk of congenital abnormality. Insulin at low physiological concentrations improved growth, whereas higher physiological concentrations were required to increase growth and development. IGF-I or IGF-II supplementation improved rat embryonic growth and development but failed to match that of the controls, indicating that other growth factors including insulin may also be required.

Abnormalities, Drug-Induced↗
PubMed1990

The effect of macromolecular rat serum fractions on conceptuses cultured in human serum: role of transferrin.

We describe the results of experiments to study the effects on rat conceptuses cultured in human serum supplemented with rat serum which was separated into high (greater than 30 kDa) and low (less than 30 kDa) molecular weight fractions by ultrafiltration. Ultrafiltered rat serum was found to lack certain growth-promoting substances which are necessary for embryonic growth and differentiation during the culture period. Culture in whole human serum or human serum supplemented with low molecular weight fraction (filtrate) results in conceptuses which grow reasonably well but are anaemic, whilst anaemia is relieved by the high molecular weight fraction. Addition of human or rat transferrin (MW 80 kD approx.) to whole human serum alleviates anaemia, an effect observed more distinctly with rat transferrin.

Anemia↗
PubMed1989

Growth of 9.5-day rat embryos in folic-acid-deficient serum.

Rat embryos (9.5-day-old) were cultured for 48 hours in serum from diet-induced folic-acid-deficient rats. Resultant embryos were frequently abnormal; many were growth retarded and exhibited a defect in the turning mechanism that inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed, and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content of the embryos to the level observed in those grown in normal rat serum. The effects of the folate deficiency could be eliminated by supplementation with normal rat serum. The data indicate that embryos have a requirement for adequate folate in order for normal growth and differentiation to take place; they also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects on maternal metabolism. This may take the form of a disturbance in the production of maternally synthesised growth factors necessary for normal embryonic development.

Animals↗
PubMed1989

Effects of low insulin levels on rat embryonic growth and development.

The risk of congenital abnormality in diabetic pregnancy is about four times that for the normal population. Past clinical studies have suggested hyperglycemia and hyperketonemia as the factors responsible for these abnormalities, with no reference to the possible effects of low insulin levels. We examine the effect of hypoinsulinemia on rat embryonic growth and development in culture while normal glucose levels are maintained. With anti-insulin antibody bound to an affinity column containing cyanogen bromide-activated Sepharose 4B beads, insulin was selectively removed from the homologous culture serum eluted down the column. A culture of rat embryos from the early head-fold stage for 50 h in insulin-depleted normoglycemic homologous serum (insulin levels 0.055-0.18 ng/ml) showed retardation of growth and development when compared with control embryos. Adding physiological amounts (10 ng/ml) of insulin back into the insulin-depleted serum subsequently restored growth level to that of control embryos. We conclude that low insulin levels, encountered in newly diagnosed diabetic pregnancy, may be instrumental in increasing the risk of congenital abnormalities.

Animals↗
PubMed1989

A method for studying rat embryonic metabolism by microcannulation of the vitelline circulation.

A novel technique which involves the cannulation of the vitelline vessels of the 11.5 days rat embryo is described; this has allowed the introduction of both macromolecular and particulate substances directly into the embryo, thus bypassing the digestive and metabolic activity of the visceral yolk sac and facilitating the study of the fate of such substances within the embryo. The method does not affect the growth of the conceptus in vitro providing that inert solutions (e.g. azo blue dye in Hank's balanced salt solution) in volumes of less than 1.0 microliter are employed. When India ink is injected, the vitelline vessels and the intraembryonic vessels are seen to be extensively perfused. Approximately 80% of injected 125I-labelled polyvinylpyrrolidone is retained by the conceptus after injection, and it is distributed mainly in the embryo and exocoelomic cavity.

Animals↗
PubMed1988

The effect of epidermal growth factor, insulin and transferrin on the growth-promoting properties of serum depleted by repeated culture of postimplantation rat embryos.

Homologous serum, when repeatedly used for the culture of postimplantation rat embryos, rapidly loses its capacity to support growth and development. Replenishment of the 'exhausted' serum with glucose and vitamins (MEM vitamin concentrate--Flow Laboratories) together with gentle dialysis to remove small molecular weight toxic metabolites (lactate etc) fails to restore the growth-promoting properties of the serum. This suggests that 'recycled' serum has been depleted of specific growth-promoting factors. Such serum that has been subjected to dialysis can be completely replenished by addition of 30% normal rat serum. It is therefore probable that the growth promoters are originally present at very low concentrations and become rate limiting when serum is recycled. Many growth factors and hormones fall into this category and it is likely that a considerable number are involved when serum is 'exhausted' by repeated use. When insulin, epidermal growth factor or rat transferrin are added to dialysed 'exhausted' serum each effects a partial restoration of growth of rat embryos.

Animals↗
PubMed1987

A stage-dependent effect of ethanol on 9.5-day rat embryos grown in culture and the role played by the concomitant rise in osmolality.

The effects of ethanol on the rat embryo undergoing organogenesis in vitro have been studied. We have shown that embryopathy is dependent on the stage of development at which explanted embryos were treated. The early neural plate (presomite) stage was highly sensitive to a 4-hour exposure of 300 mg/% ethanol, whereas embryos explanted 5 hours later, at the late head-fold (two to six somites) stage grew normally under the same conditions. We have also established that raised serum osmolality, associated with ethanol in serum, is responsible for some but not all of the teratogenic effect. This was shown by mimicking the rise in osmolality with hyperosmotic serum, containing glycerol, NaCl, or concentrated Hanks' salts. Dilution of this osmotic effect by the addition of distilled water in part reversed these embryopathic effects.

Abnormalities, Drug-Induced↗
PubMed1986

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro.

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.

2,4-Dinitrophenol↗
PubMed1986

The giant yolk sac: a model for studying early placental transport.

Nine and a half day rat embryos can be cultured for 48 hours in whole heat-inactivated rat serum using the roller culture method described by New, Coppola & Terry (1973). We have prolonged the culture period, usually by seven days. Although the embryo dies almost immediately during this extended culture period, the yolk sac continues to grow and reaches a diameter of approximately 2 cm; we have called this the giant yolk sac. The morphology of the giant yolk sac is very similar to that of control yolk sacs (17 1/2 or 18 1/2 days in vivo), the main difference being the greatly enlarged vacuolar volume in the endodermal cells of the giant yolk sac, which have been studied morphometrically. The pinocytic nature of the giant yolk sac has been demonstrated by its ability to take up colloidal gold. Its rate of uptake of 125I-polyvinylpyrrolidone in whole serum gassed with 95% O2; 5% CO2 has been shown to be similar to the rate of uptake found in control yolk sacs under the same incubation conditions. Acid phosphatase activity was found to be similar in the giant yolk sac and control yolk sacs using both histochemical and biochemical methods. Giant yolk sacs without a contained dead embryo can be produced by removing the embryonic pole of the egg cylinder prior to incubation. They exhibit all the features detailed above. Finally it is shown that the fluid from within the extra-embryonic coelom of the giant yolk sac has some capacity to support the growth and development of 9 1/2 day rat embryos when a source of bulk protein is also provided. This model, therefore, seems to be very useful for the study of transport in a placental system. Its full potential requires further study.

Acid Phosphatase↗
PubMed1984

Phagocytic uptake of latex beads by rat peritoneal macrophages: comparison of morphological and radiochemical assay methods.

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.

Animals↗
PubMed1983

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates.

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.

Animals↗
PubMed1982

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage.

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and 125I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[125I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of 125I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of Mr 120 000 was internalized by macrophages somewhat more rapidly than copolymer of Mr 46 000, but was excluded from the yolk sac.

2,4-Dinitrophenol↗
PubMed1982

Evidence for intracellular digestion of an exogenous protein by rat peritoneal macrophages.

The differential effects of lower incubation temperature on the digestion of formaldehyde-denatured 125I-labelled bovine serum albumin by intact rat peritoneal macrophages and by macrophage homogenates were studied. Digestion by intact macrophages was abolished at 20 degrees C and below, but a near-linear relationship between digestion rate and temperature between 37 degrees C and 5 degrees C was found with homogenates. Several known inhibitors of pinocytosis were found to inhibit the generation of digestion products from 125I-labelled albumin by macrophages. It is concluded that pinocytosis is an obligatory step in the catabolism of exogenous proteins by macrophages.

Animals↗