Advances in prevention and treatment of cerebral palsy.

In recent years there have been a number of advances in understanding of predisposing and protective factors in the development of cerebral palsy in infants. Multiple gestation births, maternal infection, and maternal and fetal thrombophilic conditions all predispose to the development of CP in the infant. Opportunities for prevention of CP may develop from an improved understanding of these factors and their mechanisms of operation. Similar progress has been made in the evaluation of treatments for CP and the effects of these treatments on the individual's impairment, function, and disability. Selective posterior rhizotomy and Botulinum toxin A are now widely used in the treatment of spasticity. The challenge remains to determine how effectively these promising interventions can alter long-term function and quality of life outcomes in children and adults with CP.

CAT/CLAMS: its use in detecting early childhood cognitive impairment.

The Cognitive Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS), a neurodevelopmental tool for the cognitive assessment of infants and toddlers, correlates well with the Bayley Scales of Infant Development. In 1993 the Bayley Scales were revised and the second edition published (BSID-II). This study was designed to determine how well the CAT/CLAMS correlates with the BSID-II and its utility in identifying mild and severe cognitive impairment. Sixty-eight infants and toddlers (age range = 14-48 months), referred for suspected developmental delays, were administered the CAT/CLAMS and BSID-II and the results compared. The correlation between the two instruments was strong (r = 0.89, P<0.0001). The CAT/CLAMS was sensitive (81%) and specific (85%) for detecting overall cognitive impairment (BSID-II less than 70) and was even more sensitive (100%) and specific (96%) in detecting severe cognitive impairment (BSID-II less than 50). The physician using the CAT/CLAMS formulated a clinical impression of cognitive impairment that was sensitive (95%) and specific (84%) compared with formal psychologic testing. The CAT/CLAMS correlates well with the BSID-II. It is useful for detecting and quantifying mild and severe cognitive impairment. It permits the physician to formulate an accurate clinical impression of cognitive impairment consistent with possible mental retardation.

Diacylglycerol molecular species in plasma membrane and microsomes change transiently with endothelin-1 treatment of glioma cells.

Agonist-induced intracellular signal transduction often involves activation of protein kinase C by diacylglycerol (DAG) released from membrane phospholipids by phospholipases. Using either DAG kinase or HPLC assays to quantitatively determine DAG mass, we observed a time-dependent increase in DAG accumulation upon incubation of rat C6 glioma cells with 200 nM endothelin-1 (ET-1). Total cell DAG rapidly increased by 25-35% from a basal level of 4.5 +/- 0.3 nmol/mg protein during one min of ET-1 treatment and remained constant or slightly decreased between 1 and 2 min. Thereafter, DAG increased to a maximum (1.6-fold above basal) by 5-10 min. and remained elevated to 30 min. Resolution of DAG molecular species by HPLC after incubation of cells with ET-1 revealed that accumulation of DAG species differed in total cell lysate and subcellular compartments. In plasma membrane, major DAG species increased at 1 min. followed by a decrease at 10 min. whereas in microsomes DAG species did not change at 1 min. and decreased at 10 min. Although phospholipid sources of DAG species were not identified specifically, there was preferential hydrolysis of molecular species of phospholipid for DAG production. We propose that molecular species of DAG produced at the plasma membrane may be transferred to the endoplasmic reticulum so that phospholipid resynthesis can replenish molecular species initially utilized in signal transduction.

Classification of developmental delays.

Developmental delay is frequently used to identify children with delay in meeting developmental milestones in one or more streams of development. There is no consensus on the specific definition. Developmental delay is best viewed generically as a chief complaint rather than a diagnosis. A child suspected to have delays should always be assessed in each of the major streams of development: expressive and receptive language, including social communication; visual problem solving (nonverbal cognition); motor development; neurobehavioral development; and social-emotional development. A model developed by the National Center for Medical Rehabilitation Research is used to compare existing classifications of developmental delays. This model defines the five domains in the disability process: pathophysiology, impairment, functional limitation, disability, and societal limitation. An etiology domain is added. This model is used to illustrate how existing classification systems of cerebral palsy, mental retardation, autism, and language delay draw on information from one or more domains. The model illustrates some of the conflicts between different systems. For example, most classification systems for cerebral palsy emphasize only impairment (spasticity, dyskinesias, and topography). The current definition and classification system for mental retardation focuses on functional limitations (IQ), disability, and societal limitations, ignoring pathophysiology and details of impairment. Given the complexity of neurodevelopmental disabilities, it is unlikely that a single classification system will fit all needs.

Differential expression of MARCKS and other calmodulin-binding protein kinase C substrates in cultured neuroblastoma and glioma cells.

Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H-myristoylated proteins of 50 and 40-45 kDa were observed in cell extracts heated to > 80 degrees C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced rechange of the protein on nitrocellulose. Addition of beta-12-O-tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.

Thapsigargin selectively stimulates synthesis of phosphatidylglycerol in N1E-115 neuroblastoma cells and phosphatidylinositol in C6 glioma cells.

Phospholipid metabolism was studied in N1E-115 neuroblastoma and C6 glioma cells exposed to thapsigargin, a selective inhibitor of endoplasmic reticulum Ca(2+)-ATPase that raises the cytosolic free Ca2+ concentration [Ca2+]i. Thapsigargin caused only a transient increase of [Ca2+]i (< 1 min) in N1E-115 cells similar in magnitude and duration to agonist-induced calcium release mediated by inositol trisphosphate. Sustained elevation of [Ca2+]i due to influx of extracellular calcium, as occurs in most other cell lines including C6 cells, did not occur in N1E-115 cells. Increased uptake of inorganic phosphate (Pi) associated calcium influx was observed in C6 but not in N1E-115 cells. Thapsigargin affected phospholipid synthesis in both cell lines, most likely by inhibiting phosphatidic acid phosphohydrolase as indicated by diversion of [3H]oleic acid incorporation from triacylglycerol to phospholipid synthesis and stimulation of [32P]Pi incorporation into anionic phospholipids at the expense of phosphatidylcholine synthesis. The response to increased phosphatidate/phosphatidyl-CMP availability was cell specific. Thapsigargin (> 100 nM) selectively stimulated phosphatidylglycerol synthesis 20-30-fold in N1E-115 neuroblastoma cells while phosphatidylinositol synthesis was increased < 2-fold. In contrast, phosphatidylglycerol was not affected in C6 glioma cells and phosphatidylinositol synthesis was stimulated 8-fold by thapsigargin (> 1 microM). Agonist-stimulated calcium release did not increase phosphatidylglycerol synthesis in N1E-115 cells. Thapsigargin-stimulated phosphatidylglycerol synthesis and agonist-stimulated phosphatidylinositol synthesis could occur at the same time. Similar results were obtained with TMB-8, an inhibitor of intracellular Ca2+ release that decreases diacylglycerol utilization by blocking choline uptake and phosphatidylcholine synthesis without affecting resting [Ca2+]i. Thus [Ca2+]i does not directly mediate the effects of thapsigargin, TMB-8 or agonist stimulation on anionic phospholipid metabolism. These additional effects may limit the use of thapsigargin to assess Ca(2+)-dependence of phospholipid metabolism associated with Ca(2+)-mediated signal transduction.

Clofibrate and other peroxisomal proliferating agents relatively specifically inhibit synthesis of ethanolamine phosphoglycerides in cultured human fibroblasts.

Effects of several classes of peroxisomal proliferators on peroxisomal functions, hepatomegaly, hepatocarcinogenesis and lipid metabolism have been extensively investigated in rodents. Less is known about influences of these agents, some used as hypolipidemic drugs, on various metabolic parameters in humans. We examined effects of clofibrate, di(2-ethyl-hexyl)phthalate (DEHP) and pirinixic acid (WY-14,643) on phospholipid metabolism in human fibroblasts in culture. Clofibrate inhibited incorporation of [1-14C]hexadecanol and [1-14C]linolenic acid into ethanolamine phosphoglycerides in a time- and concentration-dependent manner; labeling of plasmalogens and non-plasmalogen ethanolamine phosphoglycerides was reduced by 40-80% compared to a generalized 10-30% inhibition of labeling of other phospholipids, including phosphatidylcholine. In pulse and pulse-chase experiments, selective inhibition of incorporation of [1,2-14C]ethanolamine, compared to [methyl-3H]choline, confirmed relative specificity of inhibition of ethanolamine phosphoglycerides. Similar concentration dependence and specificity for inhibition of phospholipid turnover was observed for DEHP and WY-14,643, in both control and mutant (Zellweger and adrenoleukodystrophy) fibroblasts, in the absence of major effects on peroxisomal markers. These observations that peroxisomal proliferators specifically inhibit ethanolamine phosphoglyceride turnover in human fibroblasts should be considered when assessing the efficacy and safety of such agents as hypolipidemic drugs or when evaluating mechanisms of proliferator action at the cellular level.

CAT/CLAMS. A tool for the pediatric evaluation of infants and young children with developmental delay. Clinical Adaptive Test/Clinical Linguistic and Auditory Milestone Scale.

The American Academy of Pediatrics recommends regular developmental screening as a part of routine child health supervision. However, the pediatrician has a limited number of tools available to further evaluate a child who is found to be suspect or abnormal on a developmental screening test. The Clinical Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS) was therefore developed to provide pediatricians with a technique to assess infants and toddlers with suspected developmental delay. The CAT/CLAMS demonstrated strong psychometric properties. Concurrent validity with the Bayley Scales of Infant Development (BSID) was demonstrated in 43 children ages 12 to 19 months who were tested on three occasions with both instruments (correlation coefficient ranging between 0.63 and 0.87; P < .001). Predictive validity 6 and 12 months later was also demonstrated in this population with correlation coefficients ranging between 0.73 and 0.77, significant at the P = .001 level. Utilizing the CAT/CLAMS as part of the pediatrician's evaluation of children with developmental concerns would allow the pediatrician to compare language and nonlanguage problem-solving abilities and, therefore, aid in diagnosis and appropriate referral.

Maternal estimates of developmental age in preschool children.

OBJECTIVE: To investigate the accuracy of maternal estimates of developmental age in preschool children with suspected developmental delay. METHODS: In a sample of 139 preschool children, aged 5 to 60 months, mothers were asked before evaluation to estimate the developmental age of their child. Maternal estimates were converted to a developmental quotient (DQ) and compared with results from standardized tests of cognitive functioning, adaptive abilities, expressive and receptive language, and visual-motor skills. RESULTS: A high correlation was found (r = 0.82; p < 0.0001) between maternal-estimate DQ and actual DQ (mean of test scores). Most mothers estimated within 15% of their child's actual functioning, and 84% of mothers estimated within +/- 5 months of actual functioning. Multiple regression found no factors that would identify mothers who were more or less accurate in estimating developmental age. Maternal-estimate DQ was sensitive (83%) and specific (83%) for mental retardation. CONCLUSION: Maternal estimates provide an accurate measure of developmental functioning and could be successfully incorporated into routine developmental surveillance of preschool children.

Serine and ethanolamine incorporation into different plasmalogen pools: subcellular analyses of phosphoglyceride synthesis in cultured glioma cells.

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both PtdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [3H(G)]L-serine and [1,2-14C]ethanolamine. Specific radioactivity of PtdSer from [3H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2-4 h. Labeled plasmalogen from [3H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-14C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients.

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 14-24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was < 10% of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL (t1/2 = 10-15 h) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) or > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transport in Niemann-Pick type II disease, and may also be suitable for diagnosis and carrier detection.

Purification of two immunologically related phosphatidylinositol-(4,5)- bisphosphate phosphatases from bovine brain cytosol.

Two phosphatidylinositol-(4,5)-bisphosphate (PtdIns-(4,5)P2) phosphatase activities were isolated from a 45% saturated (NH4)2SO4 fraction of the soluble cytosol (100,000 x g supernatant) of bovine cerebral hemispheres by ion-exchange chromatography on Q-Sepharose (Q-1 and Q-2). Each was further purified on heparin-Sepharose, butyl-agarose, and/or Cibacron blue F3GA to yield products of similar specific activity (70-100 mumol/min/mg protein, 1000-2000-fold purification). Salt was required to stabilize activity and dithiothreitol was required to preserve maximum activity and to prevent or reverse aggregation that resisted disruption by mercaptoethanol and/or SDS. Monoclonal antibodies were prepared that recognized several components in the partially purified preparations. Immunoabsorption of activity by monoclonal antibodies that had been chemically cross-linked to protein A-Sepharose followed by SDS-polyacrylamide gel electrophoresis of absorbed proteins was used to identify the active components as a 155-kDa protein in Q-1 and a 115-kDa protein in Q-2. Two antibodies recognized different epitopes in the 155-kDa phosphatase. A third antibody recognized a common epitope in both phosphatases indicating that the two enzymes are related. Both phosphatases were Mg(2+)-dependent, exhibited similar kinetic properties, and hydrolyzed PtdIns(4,5)P2 but not PtdIns(4)P, phosphatidic acid, or several other phosphate monoesters. They hydrolyzed inositol (1,4,5)-trisphosphate at 30% of the rate with PtdIns(4,5)P2 and this activity co-purified with PtdIns(4,5)P2 phosphatase activity. High molecular weight PtdIns(4,5)P2 phosphatases may be precursors of lower molecular weight soluble Type II inositol polyphosphate-5-phosphatases shown to account for the PtdIns(4,5)P2 phosphatase activity in platelets (Matzaris, M., Jackson, S.P., Laxminarayan, M., Speed, C.J., and Mitchell, C.A. (1994) J. Biol. Chem. 269, 3397-3402). The three antibodies did not inhibit activity but recognized both native and denatured (Western blots) phosphatases and should be useful tools to study the distribution, structure, and regulation of the two forms of PtdIns(4,5)P2 phosphatase.

Regulation of low density lipoprotein receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities are differentially affected in Niemann-Pick type C and type D fibroblasts.

Defective regulation of intracellular cholesterol metabolism has been investigated in cultured fibroblasts from two subtypes of Niemann-Pick type II disease: the panethnic Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD). Cell extracts from NPC and NPD fibroblasts cultured in lipoprotein-deficient medium exhibited activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that were two-fold greater than in normal cells. Addition of serum resulted in only a 15% decrease in HMG-CoA reductase activity within 6 h in these cells, compared with a decrease of 80% in normal fibroblasts. The initial rate of return to maximal values for the first 6 h after removal of serum was similar in all three cell types; thereafter, the rate was faster in the mutant fibroblasts. Binding and internalization of 125I-labeled low density lipoprotein (LDL) was not decreased within 12 h of incubation of NPC fibroblasts with serum, while a decrease of 50% was observed for both NPD and normal fibroblasts over this time period. Northern blot analysis also indicated a slower decrease in steady-state LDL receptor mRNA in NPC relative to normal and NPD cells. In all three cell types, inhibition of HMG-CoA reductase with mevinolin had no effect on serum-stimulated cholesterol esterification, while inhibition of acyl-CoA:cholesterol acyltransferase with Sandoz 58-035 did not influence HMG-CoA reductase activity, indicating that defects in these regulatory mechanisms are independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical Adaptive Test/Clinical Linguistic Auditory Milestone Scale in early cognitive assessment.

Correlations between the Clinical Adaptive Test/Clinical Linguistic Auditory Milestone Scale (CAT/CLAMS) and the Bayley Scales of Infant Development--Mental Scale (BSID) were examined in 61 infants and toddlers with suspected developmental delay. Highly significant correlations were found between the two instruments. Gender, race, and gestational age did not influence the relationship between CAT/CLAMS and BSID scores. The CAT/CLAMS was both sensitive (88%) and specific (67%) for mental retardation (BSID < 70). The CAT/CLAMS correlates with the BSID and can be used as an instrument for detecting cognitive delay.

Limited metabolic interaction of serine with ethanolamine and choline in the turnover of phosphatidylserine, phosphatidylethanolamine and plasmalogens in cultured glioma cells.

Modulation of choline phosphoglyceride turnover has been investigated extensively but less is known about regulation of serine and ethanolamine phosphoglyceride synthesis and turnover. We investigated incorporation and interactions of [3H(G)]L-serine, [1,2-14C]ethanolamine and [methyl-3H]choline in cultured glioma cells. Exogenous serine did not compete with ethanolamine or choline incorporation and did not chase labeled headgroup from ethanolamine phosphoglycerides (PE); serine displaced headgroup of prelabeled phosphatidylserine (PtdSer) resulting in less labeled PtdSer for decarboxylation. In contrast, exogenous ethanolamine markedly chased labeled headgroup of non-plasmenylethanolamine phosphoglycerides (NP-PE) with less effect on plasmalogen (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) whether headgroup was derived from [3H]serine or [14C]ethanolamine. Label in chase medium was mainly ethanolamine to 12 h; phosphoethanolamine was present with longer chase (12-48 h). Choline did not compete with serine incorporation and had little chase effect on PtdSer and PE. Choline and ethanolamine competitively interacted with preference for choline. These data suggest that (1) PtdSer synthesis in cultured glioma cells may involve more than headgroup exchange; (2) PE turnover with metabolite release to medium may involve both phospholipase D and phospholipase C; (3) acceleration of PE turnover by exogenous ethanolamine primarily involves NP-PE with lesser involvement of plasmalogen; and (4) in contrast to lack of interaction between serine and other headgroup precursors, choline and ethanolamine compete primarily at uptake.

Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells.

An 80-kDa protein labeled with [3H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4 beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100-soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of beta-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, beta-TPA-stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to alpha-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.

Calcium-independent effects of TMB-8. Modification of phospholipid metabolism in neuroblastoma cells by inhibition of choline uptake.

TMB-8 [8-(NN-diethylamino)-octyl-3,4,5-trimethoxybenzoate] blocks agonist-stimulated release of Ca2+ from intracellular sites in many cell lines and is often used to distinguish between dependence on extracellular and intracellular Ca2+. In N1E-115 neuroblastoma cells, TMB-8 did not alter the resting cytosolic Ca2+ concentration in unstimulated cells, yet phospholipid metabolism was greatly affected. At concentrations of TMB-8 (25-150 microM) that inhibit Ca2+ release, phosphatidylcholine formation was inhibited, whereas synthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine was stimulated. Unlike other cationic amphipathic compounds, TMB-8 did not inhibit phosphatidate phosphatase or enzymes in the pathway from choline to phosphatidylcholine. Choline transport was the major site of action. TMB-8 was a competitive inhibitor (Ki = 10 microM) of low-affinity (Kt = 20 microM) choline transport. When added at the same time as labelled precursor, TMB-8 also decreased cellular uptake of phosphate and inositol, but not that of ethanolamine or serine. In prelabelled cells, continued uptake and incorporation of phosphate and inositol were not affected. Under these conditions phosphatidylinositol synthesis was increased 2-fold and, like the effect on phosphatidylcholine, reached a plateau at 100 microM-TMB-8. Phosphatidylglycerol synthesis increased linearly with TMB-8 concentration to 40-fold stimulation at 150 microM, suggesting a selective effect on synthesis of phosphatidylglycerol from CDP-diacylglycerol. Phosphatidylserine synthesis was also increased up to 3-fold. These Ca(2+)-independent effects limit the use of TMB-8 in studies of cell signalling that involve stimulated phosphatidylinositol and phosphatidylcholine metabolism.

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.

Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification.

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.