Dietary restriction counteracts age-related changes in cholesterol metabolism in the rat.

The effects of ageing on the metabolism of cholesterol were examined in three different organs (liver, aorta and brain) of 6-, 12- and 24-month-old male Sprague-Dawley rats. Ageing was associated with a significant increase in intracellular cholesterol esters in all three organs. Steady state mRNA levels of multidrug resistance protein (MDR) and acylCoA:cholesterol acyl transferase (ACAT), enzymes involved in cholesterol import and esterification, were also increased. By contrast, expression of mRNA for neutral cholesterol ester hydrolase (nCEH) and caveolin-1, proteins involved in cholesterol ester hydrolysis and export, were significantly reduced. Dietary restriction is the only intervention shown to extend lifespan and retard age-related declines in function in mammals. To further explore the possible correlation between changes in cholesterol esterification and ageing, we analysed cholesterol metabolism in liver, aorta, and brain of aged rats exposed to two dietary restriction regimens: intermittent (alternate-day) fasting (IF) and food intake restriction (60% of ad libitum feeding). Both dietary regimens attenuated the age-related changes in cholesterol esters and in the expression of genes involved in cholesterol metabolism. These results provide evidence that distinctive age-associated changes in intracellular cholesterol metabolism occur in rats. Furthermore, these modifications can be partially reversed by dietary restriction, a condition known to affect the ageing process. Age-related changes in cholesterol metabolism may play a role in triggering and/or aggravating senescence-related disorders characterized by altered cholesterol homeostasis.

Cell growth and cholesterol metabolism in human glucose-6-phosphate dehydrogenase deficient lymphomononuclear cells.

Atherosclerosis is an inflammatory-fibroproliferative response of the arterial wall involving a complex set of interconnected events where cell proliferation (lymphomonocytes, and endothelial and smooth-muscle cells) and substantial perturbations of intracellular cholesterol metabolism are considered to be among the main features. Glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose-monophosphate shunt pathway, is an essential enzyme involved in both cell growth and cholesterol metabolism, raising the question as to whether G6PD deficiency may have metabolic and growth implications in a deficient population. In the present study, we investigated cell growth and cholesterol metabolism in peripheral blood lymphomononuclear cells (PBMC) from G6PD-normal (n = 5) and -deficient (n = 5) subjects stimulated with lectins (phytohaemoagglutinin and Concanavalin A). G6PD activity, DNA ([3H]-thymidine incorporation) cholesterol synthesis and esterification ([14C]-acetate and [14C]-oleate incorporation), and G6PD, HMGCoA reductase and low density lipoprotein (LDL) receptor mRNA levels (RT-PCR) all increased following lectin stimulation in both normal and G6PD-deficient cells. However, these parameters were significantly lower in G6PD-deficient cells (P < 0.05). It is of interest that G6PD-deficient PBMC, which showed lower expression of G6PD and higher expression of the LDL receptor gene than normal PBMC under basal conditions, exhibited an opposite pattern after stimulation: G6PD and HMGCoA reductase being expressed at significantly higher levels in deficient than in normal cells (P < 0.05). We conclude that the reduced capability of G6PD-deficient cells to respond to mitogenic stimuli and to synthesize cholesterol esters may represent favourable conditions for reducing the risk of cardiovascular diseases.

Prenatal sonographic diagnosis of Klippel-Trénaunay-Weber syndrome with cardiac failure.

This report describes the prenatal sonographic diagnosis of a case of Klippel-Trénaunay-Weber syndrome. The sonographic appearance of this disorder was characterized by the presence of multiple distorted cystic areas involving the right leg and abdomen and cardiomegaly with early fetal heart failure. Despite the prenatal detection of the extensive cutaneous and visceral involvement, the infant died soon after birth of high-output cardiac failure and Kasabach-Merritt syndrome.

Opposite pattern of MDR1 and caveolin-1 gene expression in human atherosclerotic lesions and proliferating human smooth muscle cells.

Cholesterol esterification and smooth muscle cell (SMC) proliferation are the crucial events in the development of atherosclerotic lesions. The objective of this study was to analyse cholesterol esterification and the expression of MDR1 (multidrug resistance), ACAT (acyl-CoA:cholesterol acyltransferase) and caveolin-1 genes in atherosclerotic and healthy vascular walls, in SMCs obtained from atherosclerotic lesions and saphenous veins. Results demonstrated higher levels of cholesterol esters, ACAT and MDR1 mRNAs and lower levels of caveolin-1 mRNA in atherosclerotic segments compared to adjacent serial sections of the same artery and the corresponding non-atherosclerotic arteries from cadaveric donors. SMCs isolated from atherosclerotic plaques manifested an increased capacity to esterify cholesterol and to grow at a faster rate than SMCs isolated from saphenous veins. In addition, when SMCs from atherosclerotic plaques were cultured in the presence of progesterone, a potent inhibitor of cholesterol esterification, significant growth suppression was observed. An increase in ACAT and MDR1 expression and a concomitant decrease in caveolin-1 expression were also observed in SMCs isolated from atherosclerotic arteries as early as 12 h after serum stimulation. An opposite pattern was found when SMCs were treated with progesterone. These findings support the idea that cholesterol esterification plays a role both in early atherogenesis and in clinical progression of advanced lesions and raise the possibility that the cholesterol ester pathway might directly modulate the proliferation of SMCs.

Lipid metabolism and molecular changes in normal and atherosclerotic vessels.

OBJECTIVES: a positive correlation between cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT), multidrug resistance (MDR1) gene expression and atherosclerotic lesions has been shown in human arteries. The objective of this study was to map the expression of MDR1, ACAT genes and the cholesteryl ester content in normal, atherosclerotic and varicose human vessels. MATERIALS: vascular segments were obtained from seven cadaveric donors, 27 patients undergoing vascular surgery for severe atherosclerotic disease and 11 patients with saphenous vein varicosities. METHODS: lipid analysis and RT-PCR of MDR1 and ACAT mRNAs were performed. RESULTS: an increase in cholesteryl ester content and in ACAT and MDR1 expression was demonstrated in relation to the age in the arteries prone to atherosclerosis; this expression was maximal in arteries from symptomatic patients. In resistant arteries and in veins cholesteryl ester accumulation was rare and light, while ACAT and MDR1 expression was not related to the age of the subjects. CONCLUSIONS: the results showed that an increase in MDR1 and ACAT expression may be responsible for the accumulation of cholesteryl esters as well as for cell growth rate acceleration in vessel sites prone to atherosclerosis.

Nuchal translucency and the acceptance of invasive prenatal chromosomal diagnosis in women aged 35 and older.

OBJECTIVE: To investigate the effect of nuchal translucency screening on use of prenatal diagnosis for chromosomal abnormalities in women aged 35 and older. METHODS: Two groups of women, referred to our center for prenatal karyotype diagnosis because of maternal age, were compared: one in 1995 and the other in 1999 after the introduction of nuchal translucency measurement. Each woman received nondirective genetic counseling, and for the 1999 group, nuchal translucency results were also discussed. Risks of transabdominal chorionic villi sampling (CVS) and amniocentesis, laboratory techniques, genetic results, and local experiences were discussed. Patient's decision to undergo prenatal diagnosis, acceptance of the nuchal translucency test (in the 1999 group), and the rate of chromosomal abnormalities diagnosed by transabdominal CVS and amniocentesis, were considered. RESULTS: Two hundred twenty-one of 982 (22%) women in the 1995 group and 421 of 1386 (30%) in the 1999 group, after nondirective genetic counseling declined invasive diagnosis (P <.05). In the 1999 cohort, 1088 of 1089 (99.9%) women of appropriate gestational age had nuchal translucency measurement. Among women seen in 1995, 214 opted for transabdominal CVS (31%) and 476 (69%) for amniocentesis. Nineteen abnormal karyotypes were detected, six by transabdominal CVS and 13 (68.5%) by amniocentesis. In 1999, 266 women (29%) opted for transabdominal CVS and 650 (71%) for amniocentesis. Twenty abnormal karyotypes were detected, 13 (65%) by transabdominal CVS and seven (35%) by amniocentesis (P <.05). CONCLUSION: Knowledge of nuchal translucency could lead to a decrease in the demand for invasive diagnosis and to a more frequent diagnosis by first-trimester transabdominal CVS.

Prenatal ultrasound diagnosis of congenital cystic adenomatoid malformation of the lung: a report of 26 cases and review of the literature.

OBJECTIVES: To evaluate the sonographic appearances and prenatal natural history of congenital cystic adenomatoid malformation of the lung. METHODS: In each case a detailed examination of the thoracic lesion and a complete fetal survey was performed. The pregnancies that elected to continue were followed to term. RESULTS: A total of 26 cases were identified. The pregnancy was electively terminated in nine cases (35%). All the remaining 17 pregnancies ended in liveborn infants (100%). The lesion disappeared completely in three fetuses (18%). Of the 14 infants in whom the lesion was confirmed at birth nine required surgery in the neonatal or post-natal period. Five children did not undergo surgery. CONCLUSIONS: Conservative management appears to be an adequate medical practice in cases of isolated congenital unilateral cystic adenomatoid malformation of the lung, in the absence of hydrops and/or acute polyhydramnios.

Transient hyperechogenicity of the fetal pericardium: a case report.

An unusually echogenic fetal pericardium was visualized using ultrasound at 21 weeks' gestation. Serial prenatal examinations revealed its progressive, spontaneous resolution. Follow-up examinations of the newborn and infant failed to demonstrate any clinical or echocardiographic abnormality. The occurrence of this unusual pericardial abnormality and its transient nature should be considered during sonographic examination of the fetus.

First trimester umbilical artery pulsatility index in fetuses presenting enlarged nuchal translucency.

The aim of this prospective study was to evaluate an umbilical artery pulsatility index (UAPI) in first trimester fetuses who present enlarged nuchal translucency (NT) measurements. UAPI was measured in 121 single fetuses with NT > or =95th centile, from 10+3 to 13+6 weeks (crown-rump length (CRL) > or =38 mm - < or =84 mm). In these fetuses there were 20 trisomy 21, and six other chromosomal abnormalities (three trisomy 18 and three monosomy X). Eighty-nine cases had normal karyotype and delivered a baby without evidence of congenital malformations. Five fetuses with normal karyotype assessed antenatally were excluded from the comparison, because of evidence of congenital malformation. A fetus with normal karyotype that was spontaneously miscarried at 14 weeks' after chorionic villus sampling was also excluded. UAPI of fetuses with enlarged NT was compared with those of 65 singleton fetuses with normal NT and normal karyotype, which were used to establish our terms of reference (5th centile, median and 95th centile). UAPI of 7/20 (35%) Down syndrome and 42/89 (47%) normal karyotype fetuses presenting enlarged NT were above the median, and respectively 2/20 (10%) and 14/89 (15.7%) were above 95th centile of normal NT and normal karyotype fetuses. No significant differences were demonstrated in the UAPI values amongst normal karyotype fetuses with normal NT or normal karyotype fetuses with an enlarged NT or trisomy 21 fetuses with an enlarged NT.

Nuchal translucency in multiple pregnancies.

AIM: To evaluate the prevalence of increased nuchal translucency (NT) in multiple pregnancies and its relation to fetal karyotype and pregnancy outcome. METHODS: We measured fetal nuchal translucency (NT) in 6,338 women pregnant from 10+3 to 13+6 weeks by ultrasound and evaluated the prevalence of NT=95th centile in 115 multiple pregnancies, including 100 pairs of twins (70 dichorionic and 30 monochorionic placentas), 9 triplets, 5 quadruplets, and one quintuplet. Chorionicity, fetal karyotype, and pregnancy outcome were also evaluated in 400 singleton pregnancies. RESULTS: NT=95th centile in a single fetus was found in 10/70 cases of dichorionic twin pregnancies (14%), in two quadruplets, in 7/30 monochorionic twin pregnancies (23.3%), and in both fetuses in one dichorionic twin pregnancy. In the control group, NT=95th centile was found in 17/400 (4.2%) cases. In multiple pregnancies, two cases of trisomy 21 and one of 47, XXY were found. NT=95th centile was found in 2/2 fetuses with trisomy 21 (one dichorionic twin pregnancy and one tetrachorionic pregnancy), but not in the 47, XXY trisomy (trichorionic triplet pregnancy). A skeletal dysplasia and a Goldenhar syndrome were found among the 10 dichorionic pregnancies with increased NT. Three intrauterine deaths of both fetuses, one congenital heart disease, and a case of twin-to-twin transfusion occurred in 7 monochorionic pregnancies with increased NT. CONCLUSION: Increased NT in multiple pregnancies indicates fetuses at risk of chromosomal abnormalities and fetal malformation, and monochorionic twin pregnancies at higher risk of adverse outcome.

Assessment of risk for chromosomal abnormalities at 10-14 weeks of gestation by nuchal translucency and maternal age in 5,210 fetuses at a single centre.

OBJECTIVE: To evaluate the screening of chromosomal abnormalities by nuchal translucency (NT) measurement. METHODS: Assessment of risk for chromosomal abnormalities by NT and maternal age in 5,210 single fetuses with karyotype and outcome already known. RESULTS: Risk was > or =1 in 300 in 640 (12.2%) of all fetuses, in 575 (11.1%) of the normal fetuses, in 38 (80.8%) of the fetuses affected by trisomy 21, and in 65 (89%) of the fetuses affected by chromosomal abnormalities. Risk was > or =1 in 200 in 477 (9.1%) of all fetuses, in 418 (8.1%) of the normal fetuses, in 35 (74.4%) of the fetuses affected by trisomy 21, and in 59 (80.8%) of the fetuses affected by chromosomal abnormalities. Risk was > or =1 in 100 in 270 (5.1%) of all fetuses, in 216 (4.2%) of the normal fetuses, in 33 (70.2%) of the fetuses affected by trisomy 21, and in 54 (73.9%) of the fetuses affected by chromosomal abnormalities. CONCLUSIONS: Risk generated by NT and maternal age is effective in screening for chromosomal abnormalities.

MDR1 gene expression in normal and atherosclerotic human arteries(1).

Recent studies have shown that a membrane p-glycoprotein, encoded by MDR1 gene, is involved in the transport of free cholesterol from the plasma membrane to endoplasmic reticulum, the site of cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Moreover, results deriving from our previous studies have shown that the rate of cell proliferation was positively correlated with cholesteryl ester levels as well as with ACAT and MDR1 gene expression. In this study, lipid content and the expression of the genes involved in cholesterol metabolism such as hydroxy-methylglutaryl coenzyme A reductase (HMGCoA-R), low-density lipoprotein receptor (LDL-R), ACAT and MDR1 have been investigated in control and atherosclerotic arteries. The results have shown that the levels of cholesteryl ester increase with the age of cadaveric donors in arteries prone to atherosclerosis (abdominal aorta, superficial femoral artery) and become predominant in advanced atherosclerotic lesions. The mRNA levels of ACAT and MDR1 showed the same age correlation, reaching the highest values in atherosclerotic specimens. These results suggest that MDR1 may be involved in the accumulation of intracellular cholesterol ester levels found in atherosclerotic lesions. Moreover, the levels of HMGCoA-R, LDL-R and ACAT gene expressions progressively increased with the age of cadaveric donors; conversely, in atherosclerotic specimens, the mRNA levels of HMGCoA-R and LDL-R drastically decreased while ACAT gene expression reached its maximum. These findings suggest a reactivation of normal homeostatic regulation of cholesterol in advanced and complicated lesions.

Fetal nuchal translucency and prenatal diagnosis of beta-thalassaemia.

In Sardinia, fetal karyotyping for couples at risk for beta-thalassaemia is offered only to women >/=35 years and for specific risk of chromosomopathies. This policy is not easily accepted by the couples who incessantly request additional karyotyping. In order to select those at highest risk of chromosomal abnormalities among young women, fetal nuchal translucency (NT) thickness measurement was performed in 510 fetuses to assess the chromosomal risk before chorionic villus sampling. A risk >/=1/100 was judged positive and worthy of additional karyotyping. 126 cases interrupted the pregnancy after a result of homozygous beta-thalassaemia, hence 384 pregnancies were included in the study. 22 (5.7 per cent) fetuses were found NT positive. A total of three chromosomal abnormalities were detected. The NT test was positive in all three cases of chromosomopathies detected (100 per cent) and in 19 of 381 (4.98 per cent) normal karyotype fetuses. No features of major chromosomal abnormalities were reported among the newborns whose NT had resulted normal. These preliminary results have confirmed the efficacy of NT testing to assess the risk of trisomy 21 and other chromosomopathies and enhanced its utility in pregnancies already suited to sampling in the first-trimester for Mendelian disorders.

G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects.

In the present study we examined gene expression and glucose-6-phosphate dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and deficient subjects. The results have shown that G6PD activity strongly increases in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD gene expression were observed in leukemic cells from G6PD deficient patients compared to G6PD normal. A similar pattern of gene expression was also observed for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results support the hypothesis that G6PD deficient cell, in order to sustain their growth, must respond to the low activity of their mutant enzyme with an increase in quantity through an induction of gene expression.

Correlation between cholesterol esterification, MDR1 gene expression and rate of cell proliferation in CEM and MOLT4 cell lines.

A positive correlation between cholesterol esterification and growth rate potential was previously found in our laboratory during the growth of CEM and MOLT4 lymphoblastic cells. In the current study, we investigated whether the rates of cholesterol esters synthesis correlate with changes of acyl-CoAcholesterol acyltransferase (ACAT) mRNA levels and of other genes implied in cholesterol biosynthesis and uptake, such as 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase and low density lipoprotein (LDL) receptor. The results showed that the more rapid growing CEM cells had lower levels of expression of HMGCoA-reductase and LDL receptors compared to MOLT4. By contrast, ACAT mRNA levels were higher in CEM cells, further supporting the concept of a possible involvement of cholesterol esters in the regulation of cell growth and division. In this study, high levels of cholesterol esterification and of expression of ACAT gene were also associated with a markedly increased expression of multidrug resistance (MDR1) gene, suggesting that MDR1 activity might contribute to regulate the rate of cell growth and division by modulating intracellular cholesterol ester levels.

Synthesis and biological evaluation of a series of substituted pyrazolo[3,4-d]-1,2,3-triazoles and pyrazolo[3,4-d]oxazoles.

In view of the biological relevance of triazole-based heterocyclic structures as antifungal, antiviral, and antitumor agents, we have synthesized a series of substituted pyrazolo[3,4-d]-1,2,3-triazoles (2e-h, 2j, 4b) which we evaluated for their cytostatic and antiviral (HIV-1 included) activity and for their capability to inhibit the multiplication of various human pathogenic fungi and bacteria. Moreover, the biological activities of a few compounds, namely pyrazolo[3,4-d]oxazoles (3a-e) and pyrazolo[3,4-d]-1,2,3-triazoles (2a-d, 4a, 5), previously obtained by us but not investigated for their biological activity, were also studied. Only compounds 3a-e were endowed with a significative antiproliferative activity on the human lymphoblastoid cell line MT-4 cells. All pyrazole derivatives proved ineffective in protecting cell cultures against the HIV-1-induced cytopathogenicity, and none of the compounds was active against the bacteria and fungi tested.

Glycosidopyrroles. Part 3. Effect of the benzocondensation on acyclic derivatives: 1-(2-hydroxyethoxy) methylindoles as potential antiviral agents.

The new of 1-(2-hydroxyethoxy)methylindole derivatives 3a-i were prepared in good yields. None of them showed any significant anti-HIV activity and therefore the benzocondensation between the 2 and 3 positions of the pyrrole ring definitely reduced the weak activity found in the analogues 1a-c.

Biochemical characterization of HIV-1 reverse transcriptases encoding mutations at amino acid residues 161 and 208 involved in resistance to phosphonoformate.

Mutations at amino acid residues 161 (Q161L) and 208 (H208Y) of the reverse transcriptase (RT) have been identified in HIV-1 variants which are resistant to phosphonoformate (PFA). In the present study, we report on the biochemical properties of recombinant RTs (rRTs) carrying either one or both of the above mutations. We also report on their susceptibility to PFA and to nucleoside (NRTI) and non-nucleoside (NNRTI) RT inhibitors. Like the wild-type (wt) enzyme, mutant rRTs H208Y and Q161L/H208Y showed a preference for Mg2+ over Mn2+, whereas the Q161L rRT preferred Mn2+. The three mutant rRTs showed degrees of PFA resistance which differed according to the template-primer used, and steady-state kinetic studies revealed an inverse correlation between their degree of PFA resistance, affinity for deoxynucleoside triphosphates (dNTPs) and catalytic efficiency (kcat/Km ratio). These results indicated that HIV-1 rRTs bearing mutations at codons 161 and/or 208 had altered dNTP binding sites which led to a PFA-resistant phenotype. However, unlike the corresponding mutant viruses, which are hypersensitive to 3'-azido-3'-deoxythymidine (AZT), 11-cyclopropyl-5,-11-dihydro-4-methyl-6H-dipyridol[3,2-b:2',3',-e] diazepin-6-one (Nevirapine) and (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5, 1-jk][1,4]benzodiazepin-2(1H)-thione. (TIBO R82150), the mutant RTs Q161L and Q161L/H208Y were resistant to 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and as susceptible as the wt enzyme to Nevirapine and TIBO R82150. Overall, these results suggest that codons 161 and 208 of the HIV-1 RT gene are involved in substrate binding as well as in NRTI recognition, and provide more insights into the mechanism by which HIV-1 becomes resistant to PFA.