A Cross-Sectional Analysis of APOE Gene Polymorphism and the Risk of Cognitive Impairments in the Alzheimer's Disease Neuroimaging Initiative Study.

Background: It is inconclusive on how apolipoprotein epsilon (APOE) gene polymorphism is associated with the risk of having mild cognitive impairment (MCI) or Alzheimer's disease (AD). Objectives: To investigate how APOE genotype is associated with the risk of MCI or AD using the data collected from the Alzheimer's Disease Neuroimaging Initiative (ADNI) participants. Methods: A cross-sectional design was used to analyze the baseline data collected from the 1,720 ADNI participants. APOE gene polymorphism was analyzed on how they are related to the risk of cognitive impairments of either MCI or AD using a percent yield (PY) method. Then cognitive functions were compared among six different APOE genotypes using a two-way ANCOVA by controlling possible confounding factors. Results: The prevalence of six APOE genotypes in 1,720 participants is as following: e2/e2 (0.3%), e2/e3 (7.4%), e3/e3 (45.4%), e2/e4 (2%), e3/e4 (35%) and e4/e4 (9.9%). The e2/e2 and e4/e4 genotypes were associated with the lowest and the highest risk respectively for cognitive impairments of either MCI or AD. Further, a worse cognitive diagnosis was associated with an increasing number of APOE e4 allele in a dose dependent manner. Participants with genotype e3/e3 had a better memory measure than those with the genotype of e3/e4. Conclusions: APOE gene polymorphism is associated with different level of risks for cognitive impairments. The heterozygous genotype e3/e4 is associated with a worse memory function compared to the genotype of e3/e3. Further investigations are needed to intervene the cognitive deteriorations in those with at risk APOE genotypes.

A Descriptive Study of Musculoskeletal Injuries in Long-Haul Truck Drivers: A NIOSH National Survey.

Long-haul truck drivers are significantly affected by musculoskeletal injuries with incidence rates 3.5 times higher than the national average. Yet, little is known about injuries that affect long-haul trucks drivers. In 2010, interviewers collected data from 1,265 long-haul truck drivers at 32 truck stops across the United States. These surveys were analyzed to describe all self-reported musculoskeletal injuries. Injuries to the arm (26.3%) and back (21.1%) were the two areas most reported in the survey. Musculoskeletal injuries were most often caused by falls (38.9%) and contact with an object or equipment (33.7%) resulting most commonly in sprains/strains (60%). This large scale survey highlights the significance of musculoskeletal injuries in long-haul truck drivers and suggests the need to develop interventions to prevent injuries and improve recovery once injuries occur.

A pilot investigation on the effects of combination transcranial direct current stimulation and speed of processing cognitive remediation therapy on simulated driving behavior in older adults with HIV.

Cognitive impairments seen in people living with HIV (PLWH) are associated with difficulties in everyday functioning, specifically driving. This study utilized speed of processing cognitive remediation therapy (SOP-CRT) with transcranial direct current stimulation (tDCS) to gauge the feasibility and impact on simulated driving. Thirty PLWH (M age = 54.53, SD = 3.33) were randomly assigned to either: sham tDCS SOP-CRT or active tDCS SOP-CRT. Seven indicators of simulated driving performance and safety were obtained. Repeated measures ANOVAs controlling for driver's license status (valid and current license or expired/no license) revealed a large training effect on average driving speed. Participants who received active tDCS SOP-CRT showed a slower average driving speed (p = 0.020, d = 0.972) than those who received sham tDCS SOP-CRT. Non-significant small-to-medium effects were seen for driving violations, collisions, variability in lane positioning, and lane deviations. Combination tDCS SOP-CRT was found to increase indices of cautionary simulated driving behavior. Findings reveal a potential avenue of intervention and rehabilitation for improving driving safety among vulnerable at-risk populations, such as those aging with chronic disease.

The impact of speed of processing training on cognitive and everyday performance.

The purpose of the present investigation was to examine the impact of speed of processing training on the cognitive and everyday abilities of older adults with initial processing speed or processing difficulty. Participants were randomized to either a speed of processing intervention or a social- and computer-contact control group. Results indicate that speed of processing training not only improves processing speed, as indicated by performance on the Useful Field of View test (UFOV), but also transfers to certain everyday functions, as indicated by improved performance on Timed Instrumental Activities of Daily Living (Timed IADL). Transfer of speed of processing training to other cognitive domains was not evident. This study provides additional evidence that speed of processing training has the potential to enhance everyday functions that maintain independence and quality of life, particularly when the training is targeted toward individuals who most need it. Further study is needed to learn about the long-term effects of such training in relation to everyday abilities.

Triacylglycerol hydrolase: role in intracellular lipid metabolism.

Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions.

Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons.

To determine the relative contribution of cell bodies and distal axons to the production of acetylcholine, we used retinoic acid to induce a cholinergic phenotype in compartmented cultures of rat sympathetic neurons. When [3H]choline was given to cell bodies/proximal axons for 24 h, 98% of the radiolabel was recovered as choline, phosphocholine, CDP-choline and phosphatidylcholine, whereas only 1 to 2% of the radiolabel was incorporated into acetylcholine. Choline taken up by cell bodies and transported to axons is poorly utilized for acetylcholine biosynthesis. In contrast, when distal axons were supplied with [3H]choline, 11% of the radiolabel was recovered in acetylcholine after 24 h, the remainder being incorporated into precursors/metabolites of phosphatidylcholine. The lack of acetylcholine synthesis in cell bodies/proximal axons could not be ascribed to an absence of choline acetyltransferase activity in this region of the neurons, since the specific activity of this enzyme was similar in cell bodies/proximal axons and distal axons. The rate of choline uptake by distal axons (15.3 4.4 nmol/5 min/mg protein) was approximately 10-fold greater than by cell bodies/proximal axons (1.6 0.8 nmol/5 min/mg protein). Moreover, choline uptake into distal axons was inhibited by 74.5% by hemicholinium-3, and by 80.1% by removal of Na(+) from the medium. In contrast, choline uptake by cell bodies/proximal axons was not significantly inhibited by hemicholinium-3 or Na(+) removal. These results suggest that the majority of axonal acetylcholine is synthesized in distal axons/axon terminals from choline taken up by a high-affinity choline transporter in distal axons.

Transcription of the CTP:phosphocholine cytidylyltransferase alpha gene is enhanced during the S phase of the cell cycle.

We have studied the transcription of the CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene in C3H10T1/2 fibroblasts as a function of the cell cycle. The cells were incubated for 48 h with 0.5% fetal bovine serum. The cells were induced into the G(1) phase of the cell cycle by the addition of medium with 10% fetal bovine serum. The cells began the synthesis of DNA after 12 h. At 16 and 20 h there was an increased amount of CTalpha mRNA that coincided with an increase in the expression of CTalpha proximal promoter-luciferase constructs (-201/+38 and -130/+38). Luciferase constructs with the basal promoter (-52/+38) showed no change in activity during the cell cycle. Incorporation of [(3)H]choline into phosphatidylcholine began to increase by 8 h after the addition of serum and peaked at 18 h. The mass of phosphatidylcholine nearly doubled between 8 and 26 h after addition of serum. CT activity increased by 6 h after serum addition and was maintained until 22 h. Thus, the increase of phosphatidylcholine biosynthesis in the G(1) phase of the cell cycle is not due to enhanced transcription of the CTalpha gene. Instead increased transcription of the CTalpha gene occurred during the S phase of the cell cycle in preparation for mitosis.

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.

The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene.

A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.

Structure, expression profile and alternative processing of the human phosphatidylethanolamine N-methyltransferase (PEMT) gene.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in a series of three methylation reactions. Preliminary studies of PEMT in humans led to the cloning of three cDNAs each of which has a different 5' untranslated region (5'UTR). To determine the origin of PEMT splice variants and to investigate expression of the gene in human liver, we isolated a bacterial artificial chromosome (BAC) clone containing the full-length human gene. Each of the three unique untranslated first exons is present in a contiguous array in the gene, confirming the integrity of the cDNAs and alternative processing of PEMT transcripts. Human liver, heart and testis contain the highest levels of PEMT transcripts and of these, liver has the greatest PEMT expression. Furthermore, each of the three PEMT transcripts is present in varying abundance in liver whereas heart and testis contain only one and two transcripts, respectively. Thus, differential promoter usage in the human PEMT gene generates three unique transcripts and confers a tissue-specific expression pattern.

A role for Sp1 in the transcriptional regulation of hepatic triacylglycerol hydrolase in the mouse.

Microsomal triacylglycerol hydrolase (TGH) hydrolyzes stored triacylglycerol in cultured hepatoma cells (Lehner, R., and Vance, D. E. (1999) Biochem. J. 343, 1-10). We studied expression of TGH in murine liver and found both protein and mRNA increased dramatically at 27 days after birth. Nuclear run-on assays demonstrated that this was due to increased transcription. We cloned 542 base pairs upstream of the transcriptional start site of the murine TGH gene. Electrophoretic mobility shift assays demonstrated enhanced binding of hepatic nuclear proteins from 27-day-old mice to the murine TGH promoter, yielding three differentially migrating complexes. DNase I footprint analysis localized these complexes to two distinct regions: site A contains a putative Sp binding site, and site B contains a degenerate E box. We transfected primary murine hepatocytes with a series of 5'-deletion constructs upstream of the reporter luciferase cDNA. Positive control elements were identified in a segment containing site A. Competitive electrophoretic mobility shift assays and supershift assays demonstrated that site A binds Sp1 and Sp3. Transcriptional activation assays in Schneider SL-2 insect cells demonstrated that Sp1 is a potent activator of the TGH promoter. These experiments directly link increased TGH expression at the time of weaning to transcriptional regulation by Sp1.

Identification of transcriptional enhancer factor-4 as a transcriptional modulator of CTP:phosphocholine cytidylyltransferase alpha.

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTalpha, CCTbeta1, and CCTbeta2. The mouse CCTalpha gene promoter is regulated by an enhancer element (Eb) located between -103 and -82 base pairs (5'-GTTTTCAGGAATGCGGAGGTGG-3') upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999) Biochim. Biophys. Acta 1436, 147-165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3', was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTalpha promoter-luciferase reporter activity and CCTalpha mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTalpha promoter activity, whereas protein lacking the DNA binding domain, mTEA(-), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTalpha gene expression.

Cholesterol in the year 2000.

Cholesterol research was one of the key areas of scientific investigation in the 20th century. Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland in the first part of the century. The structure of cholesterol was completely elucidated in 1932. With the development of isotopic tracers in the 1930s studies on cholesterol biosynthesis were initiated. In 1942 K. Bloch and D. Rittenberg showed that deuterium-labeled acetate was incorporated into the ring structure and side chain of cholesterol. Another important discovery from Bloch's laboratory was that squalene was a precursor of cholesterol. In 1956, the main elements of the biosynthetic pathway became known when isopentenyl pyrophosphate was discovered as a precursor. In 1966, J. Cornforth and G. Popjak predicted that there were 16234 possible stereochemical pathways by which mevalonate could be converted into squalene. They subsequently showed which of these pathways was correct. In the 1970s and 1980s K. Bloch was able to provide intriguing evidence for an evolutionary advantage of cholesterol over lanosterol or some of the intermediates in the conversion of lanosterol to cholesterol. The last quarter of the 20th century was when M. Brown and J. Goldstein showed that the low density lipoprotein receptor was a key regulator of cholesterol homeostasis. They have also demonstrated that cholesterol balance in the cell is transcriptionally regulated via the sterol regulatory element binding protein. In the later part of the 20th century drugs were developed that effectively lower plasma cholesterol and lessen the risk of atherosclerosis and cardiovascular disease.

Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.

Murine macrophage RAW264 were investigated for their response to lipid-free apolipoproteins. Preincubation of the cells with 300 microM dibutyryl cyclic (dBc) AMP for 16 h induced specific binding of apolipoprotein (apo) A-I to the cells and apoA-I-mediated HDL formation with cellular lipids, neither of which was detected in the absence of dBcAMP. Dose-dependent changes of the apoA-I specific binding and the apoA-I-mediated cholesterol release were largely superimposable. ApoA-II also mediated lipid release after the treatment of the cells with dBcAMP and effectively displaced the apoA-I binding to the cells. In contrast, cellular cholesterol efflux to lipid microemulsion and to 2-(hydroxypropyl)-beta-cyclodextrin was uninfluenced by the dBcAMP treatment. To induce the cellular reactivity with apoA-I, the incubation with dBcAMP required at least 6 h. Actinomycin D, cycloheximide, puromycin, and brefeldin A suppressed both the induction of apoA-I-mediated lipid release and the apoA-I specific binding to the cells. Analysis of the expression level of ABC1 mRNA by using reverse transcription-polymerase chain reaction and oligonucleotide arrays revealed that ABC1 mRNA was already expressed in the dBcAMP-untreated cells, and the dBcAMP treatment for 16 h enhanced its expression 9-13-fold. We conclude that dBcAMP selectively induces apolipoprotein-mediated cellular lipid release and accordingly high-density lipoprotein generation by inducing specific binding of apolipoprotein, but does not influence diffusion-mediated lipid efflux. The cell-apolipoprotein interaction seems to depend on cellular protein biosynthesis and transport. A substantial increase in the level of ABC1 mRNA caused by the dBcAMP treatment indicates that ATP-binding cassette transporter 1, the protein product of ABC1, may directly be responsible for the interaction, but the question about the absence of the interaction with its baseline expression level remains.

The synthesis and transport of lipids for axonal growth and nerve regeneration.

Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of lipid transport within neurons are, for the most part, unknown although lipid transport via vesicles and via cholesterol- and sphingolipid-rich 'rafts' are considered likely mechanisms. Very active anterograde and retrograde transport of lipid-containing vesicles occurs between the cell body and distal axons. However, it is becoming clear that the axon need not obtain all of its membrane constituents from the cell body. For example, the synthesis of phosphatidylcholine, the major membrane phospholipid, occurs in axons, and its synthesis at this location is required for axonal elongation. In contrast, cholesterol synthesis appears to occur only in cell bodies, and cholesterol is efficiently delivered from cell bodies to axons by anterograde transport. Cholesterol that is required for axonal growth can also be exogenously supplied from lipoproteins to axons of cultured neurons. Several studies have suggested a role for apolipoprotein E in lipid delivery for growth and regeneration of axons after a nerve injury. Alternatively, or in addition, apolipoprotein E has been proposed to be a ligand for receptors that mediate signal transduction cascades. Lipids are also transported from axons to myelin, although the importance of this process for myelination is not clear.

Uptake of lipoproteins for axonal growth of sympathetic neurons.

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.

Why expression of phosphatidylethanolamine N-methyltransferase does not rescue Chinese hamster ovary cells that have an impaired CDP-choline pathway.

The mutant Chinese hamster ovary cell line (CHO), MT58, has a temperature-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), preventing phosphatidylcholine (PC) synthesis at 40 degrees C which results in apoptosis. Previous studies (Houweling, M., Cui, Z., and Vance, D. E. (1995) J. Biol. Chem. 270, 16277-16282) showed that expression of wild-type CT-alpha rescued the cells at 40 degrees C, whereas expression of phosphatidylethanolamine N-methyltransferase-2 (PEMT2) did not, even though PC levels appeared to be maintained at wild-type levels after 24 h at the restrictive temperature. We report that the failure of PEMT2 to rescue the MT58 cell line is due to inadequate long term PC synthesis. We found that changing the medium every 24 h rescued the PEMT2-expressing MT58 cells grown at 40 degrees C. This was due to the uptake and utilization of lipids in the serum. At 40 degrees C, PC levels in the wild-type CHO cells and CT-expressing MT58 cells increased over time whereas PC levels did not change in both the MT58 and PEMT2-expressing MT58 cell lines. Further investigation found that both the PEMT2-expressing MT58 and MT58 cell lines accumulated triacylglycerol at 40 degrees C. Pulse-chase experiments indicated that lyso-PC accumulated to a higher degree at 40 degrees C in the PEMT2-expressing MT58 cells compared with CT-expressing MT58 cells. Transfection of the PEMT-expressing MT58 cells with additional PEMT2 cDNA partially rescued the growth of these cells at 40 degrees C. Inhibition of PC degradation, by inhibitors of phospholipases, also stimulated PEMT-expressing MT58 cell growth at 40 degrees C. Best results were observed using a calcium-independent phospholipase A(2) inhibitor, methyl arachidonyl fluorophosphonate. This inhibitor also increased PC mass in the PEMT2-expressing MT58 cells. When the cells are shifted to 40 degrees C, PC degradation by enzymes such as phospholipases is greater than PC synthesis in the mutant PEMT2-expressing MT58 cells. Taken together, these results indicate that PEMT2 expression fails to rescue the mutant cell line at 40 degrees C because it does not maintain PC levels required for cellular replication.

Functional significance of Sp1, Sp2, and Sp3 transcription factors in regulation of the murine CTP:phosphocholine cytidylyltransferase alpha promoter.

The transcription factor Sp1 has been implicated in regulation of the expression of the murine CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene, Ctpct (M. Bakovic, K. Waite, W. Tang, I. Tabas, and D. E. Vance. 1999. Biochim. Biophys. Acta. 1438: 147;-165). We have utilized transient transfections, mutation analysis, electromobility gel-shifts, and immunoblot analysis to test the hypothesis that expression of the CTalpha gene is controlled in part by the binding of three trans-acting nuclear factors, Sp1, Sp2, and Sp3. Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains. In Sp1-mediated transcription, Sp3 acts as an activator in a dose-dependent manner and vice versa. Sp2 represses Sp1- and Sp3-driven transcription in Drosophila SL2 cells, but stimulates transcription in C3H10T1/2 mammalian cells. Our results suggest that the predominant action of Sp proteins is a direct function of local organization of three cis-acting elements in the regions A (-31/-9), B (-88/-50), and C (-148/-128). The ability of distal C (-148/-128) and proximal A (-31/-9) regions to activate or repress transcription depends upon the cellular background. The multiple binding elements at position B (-88/-50) confer a positive regulation independent of the cell context. However, the effectiveness of Sp proteins at this site is strongly governed by neighboring sites A and C. The results suggest that the level of expression of the CTalpha gene will depend on the cell type, the availability of Sp proteins, and the structure and organization of three cis-acting elements.

Inactivation of phosphatidylethanolamine N-methyltransferase-2 in aflatoxin-induced liver cancer and partial reversion of the neoplastic phenotype by PEMT transfection of hepatoma cells.

Phosphatidylethanolamine N-methyltransferase(PEMT) is an enzyme in liver that catalyzes the stepwise methylation of phosphatidylethanolamine to phosphatidylcholine, in addition to the main pathway that synthesizes phosphatidylcholine directly from choline. We have reported that PEMT is permanently inactivated in liver cancer induced by the Solt and Farber model. Here we studied, (i) whether similar changes also occur in the progression of hepatocarcinoma triggered by aflatoxin B(1) (AFB(1)) in rats; (ii) whether the hepatoma phenotype could be reversed by over-expression of PEMT2. We found that PEMT2 protein decreased in pre-neoplastic nodules and virtually disappeared in hepatocellular carcinoma induced by AFB(1) due to decreased levels of mRNA without any deletion or mutation of the DNA sequence. PEMT activity, which reflects the function of both PEMT1 and PEMT2, was lower in nodules and negligible in the tumor, consistent with its regulation at the level of gene transcription. McArdle hepatoma cells transfected with PEMT2 failed to form anchorage-independent colonies in soft agar, while the vector-transfected control line grew efficiently. Moreover, PEMT2-transfected cells were also poorly tumorigenic in vivo in athymic mice, as shown by the lower tumor incidence, the longer cancer-free-time and the lower tumor volume and weight. Together, these data indicate that the loss of PEMT function may contribute to malignant transformation of hepatocytes.

Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.

A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.