UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Trp64----nonsense mutation in the lipoprotein lipase gene.

A lipoprotein lipase (LpL) gene defect has been identified, a G----A transition at nucleotide position 446 of exon 3, resulting in a premature termination codon (Trp----stop) at amino acid residue 64. This defect was identified in a Type I hyperlipoproteinemic subject with an amino acid residue 194 defect in the other allele. Plasma lipoprotein values as well as LpL mass and activity in postheparin plasma were determined in the subjects with the residue 64 defect and in other LpL-deficient heterozygotes. LpL mass levels in both the Type I and the other subject with a 64 LpL defect were markedly reduced. This may be explained by rapid degradation of LpL protein or decreased secretion from the 64 defective allele. Alternatively, the marked reduction or absence of mass associated with the 64 defect may be due to synthesis of a severely truncated protein which escapes immunologic detection.

Short-term exposure to high dietary cholesterol in early life: arterial changes and response after normalization of plasma cholesterol.

Elevated serum cholesterol is an established risk factor for the development of atherosclerosis but the effect of high dietary cholesterol in early life on subsequent arterial response to atherogenic diet in adult life is unknown. Weanling rabbits were exposed for 6 wk to a diet containing 0.25% cholesterol, allowed to recover for 9 wk (at least 3 wk after normalization of plasma cholesterol), and subsequently rechallenged with cholesterol to determine atherogenic response. Enhanced activity of acyl-CoA-cholesterol-acyl-transferase in aorta induced by cholesterol feeding persisted even after normalization of serum cholesterol. When rechallenged with cholesterol for 3 mo, these animals displayed significantly (p less than 0.05) increased development of aortic atherosclerosis and accumulation of cholesterol esters when compared with control animals. Exposure to cholesterol in early life appears to cause persistent changes in cholesterol ester synthetic enzyme activity in aorta after normalization of plasma cholesterol and these residual effects might increase aortic response to subsequent cholesterol challenge in adult life.

Regulation of cholesterol metabolism in fetal rabbit aorta: role of amniotic fluid factors.

This study shows that amniotic fluid enhances cholesterol esterification in arterial wall, as measured by in vitro assay of acyl-CoA:cholesterol acyltransferase (ACAT) activity and by incorporation of oleic acid to cholesteryl esters in cultured fetal aortas and smooth muscle cells. This property is mostly evident in the fraction of molecular weight greater than 100,000, and it is abolished by delipidation, indicating that stimulating factor is probably lipoprotein in nature. Despite an increased cholesterol esterification by the presence of amniotic fluid in medium of cultured fetal aortas, the content of cholesterol and cholesteryl esters was much lower. The cellular structures are better preserved in explants cultured with amniotic fluid than in control animals. This study indicates that amniotic fluid contains factors that may have a pronounced effect on arterial wall during development.

A novel property of an aqueous guaraná extract (Paullinia cupana): inhibition of platelet aggregation in vitro and in vivo

Aqueous extracts of guaraná were studied in terms of effects on the aggregation of human and rabbit platelets. Guaraná extracts have anti-aggregatory and de-aggregatory actions on platelet aggragation induced by ADP or arachidonate but not by collagen. The active material was shown to be water soluble and heat resistant and appeared to be different from salicylates, nicotinic acid or known xanthines. Guaraná extracts inhibited platelet aggregation in rabbits following either intravenous or oral administration

A novel property of an aqueous guaraná extract (Paullinia cupana): inhibition of platelet aggregation in vitro and in vivo.

Aqueous extracts of guaraná were studied in terms of effects on the aggregation of human and rabbit platelets. Guaraná extracts have anti-aggregatory and de-aggregatory actions on platelet aggregation induced by ADP or arachidonate but not by collagen. The active material was shown to be water soluble and heat resistant and appeared to be different from salicylates, nicotinic acid or known xanthines. Guaraná extracts inhibited platelet aggregation in rabbits following either intravenous or oral administration.

Cholestyramine treatment in early life of low-density lipoprotein receptor deficient Watanabe rabbits: decreased aortic cholesteryl ester accumulation and atherosclerosis in adult life.

Effect of cholestyramine treatment in early life of Watanabe heritable hyperlipidemic rabbits (an animal model lacking low-density lipoprotein receptor activity) on subsequent (6 months recovery) occurrence of natural atherosclerotic lesion and arterial cholesterol metabolism was investigated. Initial cholestyramine treatment decreased both plasma total cholesterol and HDL-cholesterol levels which normalized within 4 weeks after treatment was discontinued. At 9 months of age (age of occurrence of spontaneous atherosclerotic lesions), the extent of aortic atherosclerosis in cholestyramine pre-treated animals was modestly lower (P less than 0.05), as compared to controls, with a significant (P less than 0.05) decrease in aortic cholesteryl ester content. Furthermore, at the end of the recovery period aortic activity of acyl-CoA: cholesterol acyltransferase and neutral cholesterol esterase activity was significantly (P less than 0.05) lower in cholestyramine-pretreated animals. These studies show that early cholestyramine pre-treatment in a low-density lipoprotein receptor-deficient animal model causes persistent changes which might influence cholesteryl ester accumulation and atherogenesis in adult life, even after cholestyramine treatment is discontinued.

Cholestyramine treatment in early life. Immediate and delayed effects on arterial cholesteryl ester metabolizing enzymes in the rabbit.

Feeding of cholestyramine-enriched diet to weaned normocholesterolemic rabbits resulted in: lowering of plasma cholesterol and distinctly decreased activity of aortic acyl-CoA cholesterol acyl transferase with no changes in aortic acid and neutral cholesteryl esterase activity. At 9 weeks after cessation of cholestyramine treatment enhanced activity of both aortic esterases were noted despite normalization of plasma cholesterol. No evidence for the presence of plasma factor influencing esterases activity was found in lipoprotein-free serum from cholestyramine-treated animals. These studies show that cholestyramine treatment in early life causes immediate and delayed changes in rabbit arterial cholesteryl ester metabolizing enzymes.

Ontogeny of 6-keto-PGF1 alpha synthesis in rabbit aorta and the effect of premature weaning.

A systematic study of the ontogeny of aortic 6-ketoprostaglandin F1 alpha synthesis from birth to adult life and the effect of premature weaning on this process was investigated in rabbits. Prostacyclin (PGI2) synthesis by both endogenous and exogenous arachidonic acid and its relation to aortic arachidonic acid content was determined. It was found that 1) PGI2 synthesis from endogenous arachidonic acid increased with age, whereas 2) PGI2 synthesis from exogenous arachidonic acid decreased. This correlated with a decrease in the incorporation of [14C]arachidonic acid into phospholipids with age. Aortic arachidonic acid concentration did not change from birth until 3 wk of life but increased markedly by 5 wk of age. Premature weaning caused a decrease in the synthesis of aortic PGI2 and in aortic arachidonic acid concentration initially, but the changes did not persist in later life. These studies suggest that the utilization of exogenous arachidonic acid by aorta decreases after birth perhaps due to maturity of the enzyme systems that synthesize and utilize endogenous substrates.

Coffee extracts inhibit platelet aggregation in vivo and in vitro.

The effect of aqueous coffee extracts on platelet aggregation in humans (in vitro) and rabbits (both in vitro and in vivo) was investigated. Coffee extracts were found to have anti-aggregatory effects on in vitro platelet aggregation induced by ADP or arachidonate but not by collagen. Coffee extracts were also effective after intravenous administration in rabbits. The compound(s) responsible for these effects are water-soluble, heat-resistant, appeared to be different from salicylates, and might also be due to unidentified compounds besides nicotinic acid or known xanthines. Coffee extract and selected fractions decreased the conversion of [14C]-arachidonic acid to thromboxane B2 by the platelets. These studies show that coffee extracts contain compounds which are active in inhibiting platelet aggregation, a critical step involved in thrombosis and other vascular disorders.

Human breast milk contains a factor which markedly stimulates aortic prostacyclin synthesis.

Human breast milk has been shown to contain a potent factor which markedly stimulates the synthesis of prostacyclin by rabbit aorta. The stimulatory effect of colostrum was modest when compared to mature milk. This factor appears to be protein in nature with a molecular weight of less than 10 000. The main site of action of the factor appears to be on the conversion of arachidonic acid into prostacyclin. This factor might have a role in the optimal development of prostacyclin synthetic pathways which are not fully developed at birth.

Human breast milk stimulates bile acid secretion by fetal rabbit liver in organ culture.

Fetal livers from rabbits at 30 days of gestation were grown in organ culture and the effect of human milk added to the culture medium on the ability of liver to excrete bile acids (cholylglycine) was examined. Human breast milk promoted a concentration related increase in cholylglycine accumulation in the medium. The factor(s) present in milk responsible for this effect appear to be non-protein in nature and is associated with the floating lipid fraction. Furthermore, milk enhances the integrity of liver explants, as established by light microscopy.

Ontogeny of guinea pig liver cholesterol 7 alpha-hydroxylase: unexpected inverse correlation of enzyme activity with expanding bile acid pool.

Changes in the activity of guinea pig liver cholesterol 7 alpha-hydroxylase (rate limiting enzyme of cholesterol catabolism) from birth to adult life was investigated using a microsomal acetone extraction method (to remove endogenous cholesterol). Contrary to the previously held notion, it was noted that while the total bile acid pool increased progressively with age after birth, hepatic cholesterol 7 alpha-hydroxylase specific activity declined. Neonatal hepatic cholesterol 7 alpha-hydroxylase showed an increase in enzyme activity in response to cell supernatant factors (100,000 xg supernatant) from neonatal livers, but not from adult livers.

Delayed effects of experimental maternal diabetes on plasma cholesterol level and vascular prostacyclin synthesis in the offspring.

The effect of streptozotocin-induced diabetes during pregnancy in rats on prostacyclin synthesis in aorta and heart of offsprings was investigated. Although the aortic and heart syntheses of 6-keto-PGF1 alpha in the offsprings of diabetic rats were not altered at birth, a significant rise in aorta and a decrease in the heart were evident at weaning. At weaning, offsprings of diabetic rats also show a significant rise in plasma cholesterol. These studies show that maternal diabetes might cause effects in the offspring which might become evident in later life.

High acyl-CoA cholesterol acyl transferase activity in fetal rabbit aorta: evidence for the presence of stimulating factor(s) in amniotic fluid.

The activity of Acyl CoA-cholesterol acyl transferase was markedly high in fetal aortas when compared to maternal and adult male rabbits. This activity dropped by 50% at 1 week of age. This high activity in fetal aorta a) did not appear to be due to changes in plasma cholesterol levels or to the later development of endogenous inhibitor in the aorta, but rather b) due to stimulatory factor(s) present in amniotic fluid.

Premature weaning-induced changes of cholesterol metabolism in guinea pigs.

Premature weaning in animals causes higher serum cholesterol levels and a relative inability to handle cholesterol challenge in adult life. The mechanisms responsible for these delayed effects of premature weaning are not clear. The aims of this investigation were to 1) determine the effect of premature weaning on the activity of hepatic cholesterol 7 alpha-hydroxylase and plasma cholesterol changes and 2) determine whether addition of cholesterol to the diet will restore premature weaning-induced changes. Premature weaning did not prevent the phenomenon of neonatal hypercholesterolemia noted in day 5 of normally weaned guinea pigs. At day 10, prematurely weaned animals had lower serum cholesterol when compared with normally weaned animals. Premature weaning caused (without or with cholesterol in diet) a significant decrease in the hepatic activity of cholesterol 7 alpha-hydroxylase, and this difference persisted (after 1 mo) when fed the stock diet. These studies demonstrate 1) premature weaning on day 2 does not prevent neonatal hypercholesterolemia on day 5, 2) premature weaning causes a decrease in hepatic cholesterol 7 alpha-hydroxylase activity that persists into adult life, and 3) dietary cholesterol intake in early life is unable to negate premature weaning-induced changes.

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts.

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.

Human breast milk stimulates rat liver cholesterol 7 alpha-hydroxylase activity in vitro.

Human breast milk at concentrations of (40 microliter/ml) markedly stimulates the activity of cholesterol 7 alpha-hydroxylase (rate limiting enzyme involved in cholesterol catabolism) in rat liver microsomal preparations. This activity persisted after a) cold acetone extraction (to remove cholesterol) b) dialysis and c) boiling and trypsin treatment of milk. Homogenized cow's milk and infant formula (Similac) also possessed the stimulating activity. These results suggest that milk might provide some factor(s) for the development of cholesterol catabolic process which is immature at birth.

Effect of glucocorticoid administration early in life on aortic prostaglandin synthesis and morphology in atherosclerosis-susceptible pigeons.

Effect of dexamethasone administration on aortic morphology, cholesterol content and synthesis of prostaglandins from (14C)-arachidonic acid in aorta of spontaneously atherosclerosis susceptible pigeons was examined. Dexamethasone markedly inhibited the synthesis of prostaglandin E2 and stimulated the synthesis of prostaglandin I2 in aorta. In aorta of glucocortocoid treated animals, endothelial abnormalities noted in control birds were decreased and numerous surface protrusions or 'microvilli' were noted. The possibility that glucocorticoid induced inhibition of PGE2 synthesis in pigeon aorta may contribute to improved aortic morphology is discussed.