High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity.

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.

Enhanced differentiation of HL-60 leukemia cells to macrophages induced by ciprofibrate.

Ciprofibrate, an hypolipidaemic peroxisome proliferator, induced differentiation of HL-60 cells. The effect was greatly potentiated by phorbol 12-myristate 13-acetate at a concentration where neither phorbol ester nor ciprofibrate alone had any effect on these cells. As occurs for HL-60 cell differentiation induced by high phorbol ester concentration, the ciprofibrate-induced phorbol ester-dependent differentiation of HL-60 cells proceeded through the monocytic/macrophage pathway and induced the phosphorylation of proteins with similar molecular weights suggesting that increased protein kinase C activity may be involved in the effect. The peroxisome proliferator-activated receptor (PPARalpha) transcription factor is expressed in HL-60 cells, but no changes were observed in its expression upon HL-60 cell differentiation.

Interrelationships between oxytocin and eicosanoids in human fetal membranes at term gestation: which role for leukotriene B4?

The existence of a functional paracrine loop between oxytocin and prostaglandin F2-alpha in human placental cells has been demonstrated. The present study was undertaken to investigate further the possible interrelationships between oxytocin and eicosanoids in human intrauterine tissues at term gestation. Therefore, we evaluated the effect of leukotriene B4 (LTB4) on oxytocin (OT) production by explants of fetal membranes and amnion and the effect of oxytocin on the production of LTB4 and prostaglandin E2 (PGE2) by both fetal membranes and amnion. In all cases studied (n = 25), short-term cultures of tissue explants (fetal membranes or amnion) have been carried out. The production of eicosanoids and oxytocin in culture medium was evaluated. Oxytocin measurement was carried out by radioimmunoassay following extraction of the substance with Sep Pak C18 cartridges, PGE2 and LTB4 were measured by radioimmunoassay directly in culture medium. Results show that LTB4 has no significant stimulatory effect on oxytocin production by fetal membranes or amnion tissue. On the other hand, oxytocin stimulates PGE2 release by both fetal membranes and isolated amnion, but has no effect on LTB4 production by these tissues. Taken together, these findings suggest the following conclusions: (1) a paracrine loop between LTB4 and oxytocin is lacking in fetal membranes and amnion at term pregnancy; (2) oxytocin exerts a stimulatory effect on PGE2 release by both fetal membranes and amnion; (3) the interrelationships between oxytocin and the different eicosanoids in the above tissues seem to be highly selective.

Isolation of intact organelles by differential centrifugation of digitonin-treated hepatocytes using a table Eppendorf centrifuge.

A quick subcellular fractionation procedure using differential centrifugation, which is applicable to isolated and cultured cells, is presented. This technique was developed for studying the subcellular localization of phosphorylated proteins in isolated liver cells after various stimuli, but is also applicable to many other situations. The main difference with the usual techniques is that by including digitonin in the homogenization buffer, the procedure is greatly shortened. Furthermore, because the soluble fraction is separated from the particulate fraction very early in the fractionation procedure, subcellular organelles are not exposed to phosphatases and other soluble enzymes such as esterases and proteases during the fractionation. The entire procedure is carried out in an Eppendorf centrifuge, which allows isolation of the cytosolic fraction in less than 1 min, a washed nuclear fraction in about 4 min, a mitochondrial fraction in less than 10 min, and a washed light mitochondrial L fraction in about 40 min. Judging by the behavior of marker enzymes and the morphology of the fractions, the method is highly comparable to classical procedures.

Effects of glucocorticoids and progesterone on prostaglandin E2 and leukotriene B4 release by human fetal membranes at term gestation.

The present study was undertaken to evaluate the effects of the glucocorticoid hormones betamethasone and hydrocortisone, and of progesterone on the relative production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) by explants of human fetal membranes at term gestation in the absence of labor. Tissues (n = 7) were incubated either in the presence or in the absence of the above mentioned hormones. PGE2 and LTB4 were measured in culture medium by radioimmunoassays. Glucocorticoids and progesterone did not affect PGE2 output by tissues; however, they greatly stimulated LTB4 production. Moreover, both betamethasone and hydrocortisone significantly increased the ratio of LTB4 to PGE2 formation by tissues. These results suggest that glucocorticoid hormones and progesterone might influence arachidonic acid metabolism in human fetal membranes by stimulating the production of lipoxygenase rather than cyclooxygenase substances before the onset of labor.

Overexpression of mdr2 gene by peroxisome proliferators in the mouse liver.

BACKGROUND: In mice, fibrates induce mdr2 gene expression, and its encoded P-glycoprotein in the canalicular domain of hepatocytes, as well as increasing biliary phospholipid output. It is not known whether this effect is restricted to fibrates or is a common property of peroxisome proliferators. AIMS: To test the effect of structurally unrelated peroxisome proliferators on mdr2 gene expression and biliary phospholipid output, and to explore the molecular mechanism(s) of mdr2 gene induction. METHODS: Male CFI mice were fed on a diet supplemented with several peroxisome proliferators: phenoxyacetic acid herbicides, plasticizers, acetylsalicylic acid and partially hydrogenated fish oil. RESULTS: Increased levels of mdr2 mRNAs, assessed by Northern blot analysis, were observed in the liver of mice treated with phenoxyacetic acid herbicides: 2,4,5-trichlorophenoxyacetic acid 570+/-133%, 2,4-dichlorophenoxyacetic acid 233+/-54% (p<0.005); plasticizers: di-(2-ethylhexyl)phthalate 282+/-78%, di-(isoheptyl)phthalate 163+/-40%, phthalic acid dinonyl ester 225+/-48% (p<0.01); and partially hydrogenated fish oil 372+/-138% (p<0.005). P-glycoprotein traffic ATPase content increased in the canalicular domain of hepatocyte of mice treated with the herbicide 2,4,5-trichlorophenoxyacetic acid and with partially hydrogenated fish oil (108% and 87%, respectively, p<0.05) as well as biliary phospholipid output (106% and 74%, respectively, p<0.05). In 2,4,5-trichlorophenoxyacetic acid-fed mice we found five-fold increase on mdr2 transcription rate, assessed by nuclear run-off assay. CONCLUSIONS: Peroxisome proliferators induce mdr2 gene, its encoded P-gp in the canalicular domain of hepatocytes and increase biliary phospholipid output. The modulation of mdr2 gene might be part of the pleiotrophic response of peroxisome proliferation in mice liver and seems to be regulated mainly at a transcriptional level.

Apolipoprotein E polymorphism in patients with acute pancreatitis.

We have shown that patients with previous acute pancreatitis (AP) may have an abnormal catabolism of chylomicron remnants (CMR). Because apoprotein E (Apo E) genetic polymorphism has an important influence on CMR clearance, we compared frequency distribution of Apo E phenotypes in 52 patients with AP, 109 patients with gallstones, and 110 control subjects. Apo E phenotypes were detected by isoelectric focusing and immunoblotting. After adjusting for differences in age and gender, fasting triglyceride level was comparable between the study groups. The frequency distribution of Apo E phenotypes was not different between the three study groups and it was in Hardy-Weinberg equilibrium. The gene frequency for Apo E2 was 0.212, 0.273, and 0.243 in AP, gallstone, and control group, respectively. For Apo E3 it was 0.701, 0.627, and 0.674, and for Apo E4 0.090, 0.100, and 0.083 in the same groups, respectively. Differences were not statistically significant (chi 2). In conclusion, the abnormal catabolism of CMR in patients with AP is not attributable to Apo E polymorphism. An alternative explanation may be sought in the activity of the recently identified hepatocytic Apo E receptor [LDL-related receptor protein (LRP)].

[Serum beta hCG levels and transvaginal echography in the early phases of pregnancy]. / Beta hCG plasmatica ed ecografia transvaginale nelle fasi precoci di gravidanza.

One hundred thirty two patients at an early gestational age were monitored every other day to establish beta hCG levels at which the gestational sac, the yolk sac and fetal heart motion can be sonographically detected. These structures were observed at significantly lower beta hCG levels by means of the vaginal probe in comparison with the abdominal one. In normal pregnancy a gestational sac of 1-3 mm was detected at a mean hCG level of 1150 UI/l (range 800-1500); the yolk sac was detected at a mean hCG level of 6000 UI/l (range 4500-7500); fetal hart motion was visible at a mean hCG level of 10,425 UI/l (range 8650-12,200). The yolk sac and fetal heart motion were always detected when the gestational sac was greater than 11 and 17 mm respectively. A practical method to evaluate early first trimester pregnancy is suggested.

[Umbilical flowmetry: effect of measurement sites on the results]. / Flussimetria ombelicale: influenza del sito di misurazione sui risultati ottenuti.

Pulsed Doppler duplex sonography was performed longitudinally on 11 uncomplicated singleton pregnancies from 20 to 41 week's gestation (87 measurements). Blood flow velocities were recorded at 3 locations: abdominal insertion site (O), in the free floating part (C), placental insertion site (PL). The objective of the study was to determine whether the C site S/D ratio, the quickest and easiest to perform, relates to the other sites. The S/D ratio measurements fell from the near abdomen (O) to the free floating part (C) to the placental insertion site (PL) between 25-35 week's gestation. Statistical analysis, including analysis of variance for repeated measures and student t test, showed no significant difference in S/D ratio among the locations before 25 and after 35 week's gestation. The S/D ratio at the O site was significantly higher than at the PL site between 25-35 week's gestation; the C site was not significantly different from the PL site.