Type C Niemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing

To investigate biochemical heterogeneity within Niemann-Pick type C disease (NPC), the two most characteristic abnormalities, namely (1) kinetics of LDL-stimulated cholesteryl ester formation and (2) intravesicular accumulation of LDL-derived unesterified cholesterol, evaluated by histochemical filipin staining, were studied in cultured skin fibroblasts from a population of 125 NPC patients. Profound alterations (esterification rates less than 10% of normal, very numerous and intensely fluorescent cholesterol-filipin granules) were demonstrated in 86% of the cases, depicting the 'classical' NPC phenotype. The remaining cell lines showed a graded less severe impairment and more transient delay in the induction of LDL-mediated cholesteryl esterification, along with an attenuated accumulation of unesterified cholesterol. In particular, cells from a small group (7%) of patients, which have been individualized as representative of a 'variant' phenotype, showed only slight alterations of esterification, restricted to the early phase of LDL uptake and undistinguishable from those in heterozygotes. In these cells, an abnormal cytochemical distribution of LDL-derived cholesterol, although moderate, was still evident provided rigorous experimental conditions were followed. A third, less clearly individualized group (7%), differing from the classical phenotype mostly by higher rates of cholesteryl ester formation, has been designated as an 'intermediary' phenotype to reflect a more difficult diagnosis of such patients. These findings have an important bearing with regard to diagnosis and genetic counselling, although the significance of such a phenotypic variation in terms of genetic heterogeneity has still to be demonstrated. A given biochemical phenotype was however a constant observation within a family (14 pairs of siblings tested so far). The unique feature of LDL-cholesterol processing alterations in NPC has been further established from comparative studies in Wolman disease and I-cell disease, showing normal or different intracellular distribution of unesterified LDL-derived cholesterol in the latter disorders. Correlation between biochemical and clinical NPC phenotypes was only partial, but a correlation between the severity of alterations in cholesterol processing and sphingomyelin catabolism could be established.     

Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein

Cholesteryl ester (CE) accumulation in arterial wall macrophages (foam cells), mediated by the intracellular enzyme acyl coenzyme A:cholesterol acyl transferase (ACAT), is a prominent feature of atherosclerotic lesions. However, native low density lipoprotein (LDL) does not cause activation of ACAT or CE accumulation in cultured mouse peritoneal macrophages despite both substantial LDL uptake and degradation and the presence of ACAT in these cells. We now report that when protein synthesis is inhibited in mouse peritoneal macrophages by treatment with cycloheximide, puromycin, or actinomycin D, native LDL-induced whole-cell ACAT activity and CE accumulation is 10-fold higher than that seen in LDL-treated control cells. The enhancement of ACAT activity was seen 4 h after the addition of cycloheximide, and ACAT activity returned to control values 4 h after the withdrawal of cycloheximide. Postnuclear supernatants and microsomes from cycloheximide-treated mouse peritoneal macrophages also had higher ACAT activity than microsomes from control cells, but the relative enhancement (maximum 3.3-fold) was less than that seen when ACAT was assayed in the intact cell. In contrast to the situation with mouse peritoneal macrophages, cycloheximide treatment of J774 macrophages, which under normal conditions display high ACAT activity and CE accumulation in the presence of native LDL, did not result in further enhancement of either ACAT activity or LDL-induced CE accumulation. From these data we postulate that mouse peritoneal macrophages have a short-lived protein that inhibits ACAT-mediated cholesterol esterification which is responsible for their lack of ACAT response and CE accumulation in the presence of native LDL. The explanation for high ACAT activity and LDL-induced CE accumulation in J774 macrophages may be that these cells lack the putative mouse peritoneal macrophage cholesterol esterification inhibitor.     

A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.     

Evidence that prefibrotic myelofibrosis is aligned along a clinical and biological continuum featuring primary myelofibrosis

Purpose

In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.

Patients and Methods

We investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.

Results

As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.

Conclusion

Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.     

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.     

Adenylate kinase isoenzyme patterns in tissues of people of different phenotype

Three distinct isoenzyme sets have been demonstrated for adenylate kinase (AK) from human tissues by the technique of starch gel electrophoresis. In adult and fetal muscle, liver, kidney, brain, spleen, lung, and leukocytes the same set was found, though individual bands had different intensities. In adult red cells and in fetal red cells separate and distinctive isoenzyme sets were observed. However, in all tissues, adult and fetal, the phenotype of an individual was recognizable by the presence or absence of a characteristic cathodal band. The apparent anomaly of the same phenotype being expressed in different isoenzyme sets was resolved by the demonstration that in adult and fetal red cells AK complexed with hemoglobin. The implications of this complex are briefly discussed.Supported by grants from the Scottish Hospital Endowments Research Trust (HERT 309) and the Muscular Dystrophy Group of Great Britain.     

Evaluation of routine prenatal ultrasound examination in detecting fetal chromosomal abnormalities in a low risk population

Prenatal diagnosis performed by fetal ultrasound scan is now a routine part of antenatal care in many countries. We have used our registry of congenital malformations to determine how many fetal anomalies and consequently how many chromosomal abnormalities are detected by this procedure. In our region, evaluation of prenatal diagnosis of chromosomal abnormalities in women of 38 years and younger (chromosomal prenatal diagnosis is offered to women 38 years) with no personal or familial history of chromosomal anomaly was performed in 119 099 consecutive pregnancies of known outcome from 1980 to 1987. At least one ultrasonographic examination seeking congenital malformations was performed in more than 95% of the pregnant women studied. The total number of chromosomal anomalies during the study period was 199, 123 of these being Down syndrome. Only 41 (34.5%) of the 119 fetuses with chromosomal abnormalities and congenital malformation examined had been found to have a malformation at ultrasound examination. This low sensitivity was different for the diverse chromosomal abnormalities. Only 10 out of the 54 fetuses with Down syndrome and malformations (18.5%) were detected and only 3 out of 24 (12.5%) atrioventricular canal defects in those trisomie 21 patients were detected. Only 5 out of 11 (45.4%) fetuses with trisomy 13, 13 out of 26 (50.0%) fetuses with trisomy 18, 7 out of 12 patients with monosomy X (58.3%) and 6 out of 27 (22.2%) fetuses with other chromosomal abnormalities were diagnosed. Moreover, the time of detection of these anomalies was early enough to allow amniocentesis and termination of pregnancy in the case of a chromosomal abnormality in only 15 out of these 41 patients, including 7 cases of cystic hygroma in fetuses with monosomy X. This low sensitivity is not the result of the quality of the ultrasound equipment. It may be explained by the inadequate qualification of some operators and by the insufficient duration of the routine examination. In conclusion, our study has shown that the sensitivity of the detection of chromosomal abnormalities by routine prenatal ultrasound screening is low. Other screening methods are needed.     

Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

A central feature of Niemann-Pick Type C (NPC) disease is sequestration of cholesterol and glycosphingolipids in lysosomes. A large phenotypic variability, on both a clinical as well as a molecular level, challenges NPC diagnosis. For example, substantial difficulties in identifying or excluding NPC in a patient exist in cases with a "variant" biochemical phenotype, where cholesterol levels in cultured fibroblasts, the primary diagnostic indicator, are only moderately elevated. Here we apply quantitative microscopy as an accurate and objective diagnostic tool to measure cholesterol accumulation at the level of single cells. When employed to characterize cholesterol enrichment in fibroblasts from 20 NPC patients and 11 controls, considerable heterogeneity became evident both within the population of cells cultured from one individual as well as between samples from different probands. An obvious correlation between biochemical phenotype and clinical disease course was not apparent from our dataset. However, plasma levels of HDL-cholesterol (HDL-c) tended to be in the normal range in patients with a "variant" as opposed to a "classic" biochemical phenotype. Attenuated lysosomal cholesterol accumulation in "variant" cells was associated with detectable NPC1 protein and residual capability to upregulate expression of ABCA1 in response to LDL. Taken together, our approach opens perspectives not only to support diagnosis, but also to better characterize mechanisms impacting cholesterol accumulation in NPC patient-derived cells.     

The implications of 7-dehydrosterol-7-reductase deficiency (Smith-Lemli-Opitz syndrome) to neurosteroid production

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation/mental retardation syndrome with an estimated incidence among individuals of European ancestry of 1 in 20000 to 1 in 30000. It is caused by inactivity of the enzyme 7-dehydrosterol-delta(7)-reductase, which catalyses the terminal transformation in cholesterol synthesis. Patients show not only an increased level of 7-dehydrocholesterol in blood and tissues, but also increased 8-dehydrocholesterol because of the presence of an active delta(8)-delta(7) isomerase. A major consequence of these biochemical abnormalities is the alteration of normal embryonic and fetal somatic development causing postnatal abnormalities of growth, learning, language and behavior. While deficient cholesterol during early development is the primary cause of central nervous system (CNS) abnormalities and retardation, we questioned whether neurosteroids could also be involved since they can have a profound influence on behavioral characteristics. Disordered neurosteroidogenesis would be expected in SLOS and could be caused by a deficiency in classical neurosteroid synthesis secondary to cholesterol deficiency, or by synthesis from 7- and 8-dehydrocholesterol of novel neurosteroids with delta(7) or delta(8) unsaturation which may have altered activity compared with conventional neurosteroids. In particular we sought analogues of dehydroepiandrosterone sulfate, pregnenolone sulfate and the pregnanolone epimers. We targeted urine from post-pubertal females, as this type of sample would be most likely to yield identifiable amounts of the pregnanolone metabolites of progesterone. Analysis by GC/MS of urinary steroids excreted by post-pubertal females confirmed the presence of neurosteroid-like compounds in SLOS patient's urine. Even though the new neuroactive steroids identified were unlikely to have been formed in the brain, it is likely that mechanisms for their synthesis are operable in this organ.     

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.     

Early onset pre-eclampsia: recognition of underlying renal disease.

A follow up study of 84 patients with early onset pre-eclampsia (before 37 weeks'' gestation) showed a high prevalence of underlying renal disease. Renal abnormalities were found in 33 of the 49 primiparas (67%) and in 22 of the 35 multiparas (63%). Two thirds of the multiparas with pre-eclampsia before 37 weeks with a diagnosis of either essential hypertension or renal disease had recurrent pre-eclampsia. Maternal morbidity and fetal mortality were greater in the group with early onset pre-eclampsia than in a group with late onset disease. Idiopathic pre-eclampsia occurred in 10% of primiparas in the early onset group, whereas it was the main condition in over three quarters of primiparas in the late onset group. A presumptive diagnosis of idiopathic pre-eclampsia is likely to be correct only in primiparas who develop the disease after 37 weeks of pregnancy; in all other cases careful search will almost certainly detect an underlying abnormality, predominantly renal.     

Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy

Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease.     

Examination of fetuses after induced abortion for fetal abnormality.

OBJECTIVE--To determine the accuracy of midtrimester diagnosis of fetal abnormality by examination and investigation of fetuses after induced abortion. DESIGN--Prospective study over five years of fetuses aborted in the midtrimester because of abnormalities detected by ultrasonography and amniocentesis. Techniques included a full external examination by a clinical geneticist with experience in dysmorphology and other investigations including necropsy. SETTING--Regional genetic centre. PARTICIPANTS--Clinicians working within the North Western region who wished to use the service offered. RESULTS--133 Fetuses were aborted because of abnormalities detected on ultrasonography and 115 because of abnormal findings on amniotic fluid analysis. In a further two cases fetal abnormality was diagnosed by molecular genetic and biochemical techniques. Among the fetuses with abnormal scans the pretermination diagnosis was changed or refined in a way which affected genetic counselling in 53 of 133 cases. Among the 115 fetuses diagnosed as abnormal by amniocentesis the pretermination diagnosis was confirmed in 112 cases and altered in three. CONCLUSION--Fetuses aborted because of abnormalities detected by screening should be examined by suitably experienced clinicians, both for accurate genetic counselling of the families and for quality control of the tests employed.     

Transport of cholesterol across a BeWo cell monolayer: implications for net transport of sterol from maternal to fetal circulation

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).     

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

Congenital high airway obstruction syndrome

Congenital high airway obstruction syndrome (CHAOS) is a very rare fetal malformation caused by obstruction of fetal airway because of laryngeal or tracheal atresia, subglottic stenosis, laryngeal cyst or laryngeal web. The prenatal diagnosis is inferred from secondary changes such as enlarged, hyperechogenic lungs, ascites and/or hydrops, flattened or everted diaphragms, dilated distal airways and mediastinal compression. There are only few cases of long-term survival described in literature. We present the case of fetus with such secondary changes diagnosed during routine ultrasound evaluation in 20 weeks' gestation. There were no other abnormalities and the kariotype was normal. In 26 weeks' gestation fetal hydrops appeared and subsequent polyhydramnios occurred in 28 weeks' gestation. The patient was planned for EXIT procedure during labor in experienced in CHAOS cases center. In 29 weeks' gestation the premature rupture of membranes and regular uterine contractions occurred and we've performed cesarean section. A multidisciplinary team of neonatologists, laryngologists and pediatric surgeons made their efforts to save the newborn, but there was complete laryngeal atresia and tracheal agenesia and immediate tracheostomy was impossible. The most important about CHAOS are early diagnosis, detailed fetal assessment and an adequate postnatal intervention for establishing fetal airways.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.     

Ontogenesis of cholesterol side-chain cleavage activity in the ovine adrenal during late gestation.

The present study examined the activity of the cholesterol side-chain cleavage system, and the amount of cytochrome P450scc in adrenal glands of sheep fetuses and newborn lambs as well as the in vitro regulation of these parameters. Freshly isolated fetal adrenal cells incubated in the presence of 1 mM 8Br-cAMP or 25 microM 22R-OH cholesterol, produced 4- to 5-fold less pregnenolone than neonatal cells under similar conditions. Likewise, pregnenolone production by isolated fetal adrenal mitochondria was lower than that of neonatal mitochondria when endogenous cholesterol was used as a substrate or when 22R-OH cholesterol was added to the incubation medium. Also, the amount of P450scc, determined by immunoblot, was lower in fetal mitochondria than in neonatal mitochondria. In culture, ACTH, despite enhancing both the production of pregnenolone and the incorporation of [14C]acetate in cholesterol and its end-products by fetal adrenal cells, neither increased the amount of pregnenolone formed from 22R-OH cholesterol nor the amount of immunoreactive P450scc. By contrast, during the first 48 h of culture under standard conditions, there was a "spontaneous" increase in the activity of P450scc which reached values observed in neonatal adrenal cells. Such a development was inhibited when 5% ovine fetal serum was added to the culture medium. These results reinforce the view that in the ovine fetal adrenal gland, the development of P450scc is not ACTH-dependent but involves most probably a decrease in inhibitory factors present in fetal blood.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.