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Biomedical subjects

G Gil

Publications and source records attributed to G Gil.

At least 19 recordsLinked to original sources
PubMed2005

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells.

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.

Bile Acids and Salts↗
PubMed2005

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

Adenoviridae↗
PubMed2005

Single-nucleotide polymorphisms in the p53 pathway.

A cell culture assay has been developed that detects and validates single-nucleotide polymorphisms (SNPs) in genes that populate the p53 pathway. One hundred thirteen EBV-transformed human B-lymphocyte cell lines obtained from a diverse population were employed to measure the apoptotic response to gamma radiation. Each cell line undergoes a reproducible, characteristic frequency of apoptosis, and the response of the population forms a normal distribution around a median of 35.5% apoptosis with a range from 12% to 58% apoptosis. Polymorphisms in the AKT1 and Perp genes significantly affect the frequency of apoptosis. The assay can detect both racial and sexual dimorphisms in these genes and has the ability to demonstrate epistatic relationships within the p53 pathway. The cell lines used in this assay provide biological materials to explore the molecular basis of the polymorphisms.

Black or African American↗
PubMed2002

Theoretical efficiency of a televisiting service for home care support.

We have made a theoretical study of the potential for televisiting. All journeys made by the home care support team in an area of Madrid were reviewed to study the possibility of using televisiting. During the year 2000 the team provided an average of seven home visits a day. The home visits took 30 min on average, and the journey time was 30 min for urban areas and 45 min in the suburbs. We estimated that two of each day s visits could be carried out by telemedicine, with a mean duration of 15 min each. A model was used to calculate the resource demands and the potential cost-savings of televisiting compared with traditional home care. Using televisiting, 62% more suburban oncology patients could be managed and a cost reduction of euro;6 per visit could be achieved after 10 weeks.

Aged↗
PubMed2001

Suppression of sterol 12alpha-hydroxylase transcription by the short heterodimer partner: insights into the repression mechanism.

Cholesterol conversion to bile acids is subject to a feedback regulatory mechanism by which bile acids down-regulate their own synthesis. This regulation occurs at the level of transcription of several genes encoding enzymes in the bile acid biosynthetic pathway. One of these enzymes is sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase), the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Previous studies from this laboratory showed that fetoprotein transcription factor (FTF) is required for 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Here, we report that the short heterodimer partner (SHP) suppresses 12alpha-hydroxylase promoter activity via an interaction with FTF. Hepatic nuclear factor-4 (HNF-4) binds and activates the 12alpha-hydroxylase promoter and is required for 12alpha-hydroxylase promoter activity. Although HNF-4 interacts with SHP, it is not involved in SHP-mediated suppression of 12alpha-hydroxylase promoter activity. FTF and not HNF-4 is the factor involved in regulation of 12alpha-hydroxylase promoter activity by bile acids through its interaction with SHP. Finally, interaction of SHP with FTF displaces FTF binding to its sites within the 12alpha-hydroxylase promoter. These results provide insights into the mechanism of action of bile acid-mediated regulation of sterol 12alpha-hydroxylase transcription.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2001

Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo.

BACKGROUND & AIMS: The rate of 12alpha-hydroxylation of bile acid intermediates is believed to determine the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) biosynthesis and the overall hydrophobicity of the bile acid pool. The aim of this study was to determine the effects of the level of expression of sterol 12alpha-hydroxylase (CYP8b1) and cholesterol 7alpha-hydroxylase (CYP7a1) on rates of CA biosynthesis and bile acid pool composition. METHODS: Expression of CYP8b1 and CYP7a1 was accomplished through infection of primary rat hepatocytes (PRH) or intact male SD rats with replication-defective recombinant adenoviruses encoding either CYP8b1 or CYP7a1. RESULTS: Increased expression of CYP7a1 over basal levels in PRH dramatically increased bile acid biosynthesis (586% +/- 82%, P < 0.001) but did not alter the ratio of CA to CDCA. Conversely, increased expression of CYP8b1 in vitro had no significant effect on the rates of total bile acid synthesis but significantly increased (4.1-fold) the rates of CA biosynthesis, resulting in an increase in the CA-CDCA ratio from 1:6.6 to 2.8:1. In whole rats, increased CYP8b1 expression over basal levels markedly increased the CA in the bile acid pool from 36% +/- 3.4% to 50% +/- 2.9% in 5 days. CDCA and its muricholic acid derivatives decreased from 64% +/- 3.4% to 50% +/- 2.9%. CONCLUSIONS: Increased expression of CYP8b1 led to a marked increase in CA biosynthesis both in PRH and in whole animals. CYP8b1 is capable of 12alpha-hydroxylating bile acid intermediates from both the classic and acidic pathways.

Animals↗
PubMed2000

Role of FXR and FTF in bile acid-mediated suppression of cholesterol 7alpha-hydroxylase transcription.

Bile acid biosynthesis is subjected to feedback regulation whereby bile acids down-regulate their own synthesis. The major point of this regulation is at the level of cholesterol 7alpha-hydroxylase (7alpha-hydroxylase), which controls bile acid output from the classic pathway. This regulation is at the level of transcription of the gene. Two bile acid response elements have been localized within the 5'-flanking region of the rat gene and these elements overlap three nuclear receptor binding sites for hepatocyte nuclear factor (HNF-4), liver X receptor (LXR) and alpha(1)-fetoprotein transcription factor (FTF). Recently it has been shown that bile acids are physiological ligands for the farnesyl X receptor (FXR), which suggested that FXR could function by binding to one of the three nuclear receptor sites to mediate regulation of 7alpha-hydroxylase transcription by bile acids. In this study we show that FXR is indeed a crucial factor for bile acid-mediated regulation, but that it functions without binding to DNA. Furthermore, we also demonstrate that neither the LXR nor the HNF-4 sites are involved in bile acid-mediated regulation of 7alpha-hydroxylase transcription. Most importantly, we show that the FTF site is essential for regulation of 7alpha-hydroxylase by bile acids, similar to what we have recently demonstrated for another gene of the bile acid biosynthetic pathway, the sterol 12alpha-hydroxylase gene. These studies demonstrate the crucial role of FTF in the expression and regulation of a critical gene in the bile acid biosynthetic pathways.

Animals↗
PubMed2000

Alpha 1-fetoprotein transcription factor is required for the expression of sterol 12alpha -hydroxylase, the specific enzyme for cholic acid synthesis. Potential role in the bile acid-mediated regulation of gene transcription.

Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways. Sterol 12alpha-hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12alpha-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12alpha-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind alpha(1)-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12alpha-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways.

Animals↗
PubMed2000

[Self-assessment by patients of impairment caused by obstructive sleep apnea syndrome].

BACKGROUND AND OBJECTIVE: The compliance of the nasal continuous positive airway pressure treatment (nCPAP) of the obstructive sleep apnea syndrome (OSAS) depends on the patients' awareness and improvement of the symptoms. Therefore, we studied the relevance of OSAS in different aspects of the quality of life and important aspects of daily life (driving performance, working place). METHODS: 352 patients with OSAS filled in a questionnaire of sociodemographic data and impairment of quality of life. The relevance of OSAS was evaluated with polysomnography and parameters of daytime sleepiness. RESULTS: The number of women was lower than expected from community related data. The apnea/hypopnea index was 29.9 +/- 23.4/h at baseline measurement and was reduced to 8.8 +/- 9.6/h with nCPAP (p < 0.001). The arousal index (baseline 26.6 +/- 15.3/h) fell with treatment significantly (17.7 +/- 9.2 Arousals/h, p < 0.001). The error rate in the driving simulator test was 7.8 +/- 8.9% (normal value 5.75 +/- 1.4). It was normalised with nCPAP (5.8 +/- 9.5, p < 0.001). 42.3% of patients reported about sleep apnea related symptoms for more than five years before diagnosis. The patients rated their physical fitness on a scale (0 to 6) at 2.9 +/- 1.3. It increased significantly with treatment to 3.4 +/- 1.3 an (p < 0.001). The mental activity improved significantly, too. The daytime sleepiness improved from 3.4 +/- 1.5 to 3.1 +/- 1.5 (p < 0.001). 32.3% of the patients reported that they fell asleep while steering at least once in the last month, 13.0% reported about more than five episodes/month. 5.3% of the patients thought their job in danger because of OSAS. 65.1% felt their performance in job to be impaired. CONCLUSION: OSAS impairs patients in different aspects of daily life, especially in the working place. The latency between the onset of symptoms and diagnosis is longer than expected. NCPAP treatment improves the symptoms of OSAS significantly both in criteria of polysomnography and self-assessment of the patients.

Activities of Daily Living↗
PubMed2000

Biochemical and molecular characterization of a laccase from Marasmius quercophilus.

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the "blue copper oxidase" family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75 degrees C, and the K(m) was 7.1 microM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

Agaricales↗
PubMed2000

Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17.

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.

Agaricales↗
PubMed2000

A test for the determination of sustained attention in patients with obstructive sleep apnea syndrome.

BACKGROUND AND OBJECTIVES: To investigate the parameter daytime sleepiness in patients with the sleep apnea syndrome (SAS), a test for measurement of sustained attention was developed. The present studies were performed on volunteers undergoing preemployment medical examinations and SAS patients to determine the extent to which test results are in agreement with the symptoms of SAS and traffic accident reports, and also with daytime sleepiness, and whether learning or therapeutic effects can be seen with repeated tests prior to and following treatment with nasal continuous positive airway pressure (nCPAP). METHODS PARTICIPANTS: 125 healthy volunteers, and two groups of 28 SAS patients each. DESIGN: Study A: The volunteers underwent a single attention test and completed a questionnaire concerned with traffic accidents and symptoms of sleep-related breathing disorders. Study B: SAS patients underwent two attention tests before treatment. Study C: SAS patients underwent one attention test before and one after nCPAP therapy. RESULTS: Study A: The error rate in volunteers without symptoms of sleep-related breathing disorders (51 persons) was 4.7 +/- 4.3% (number of errors 14.1 +/- 12.9), 95% CI: 1.2 (number of errors 3.6). No dependence of the error rate on age, BMI or sex was found. In persons with a history of apneic events (n = 10), the error rate was 10.6 +/- 10.0% (number of errors 31.8 +/- 30), in those with more than two accidents during the last 5 years (n = 4), it was increased to 15.3 +/- 9.7% (number of errors 45.9 +/- 29.1). Study B: Among SAS patients, no significant learning effect was seen, and prolongation of the test duration beyond 30 min had no effect on the test results. Study C: The error rate improved significantly with nCPAP [10.6 +/- 13.5 vs. 6.4 +/- 8.9% (number of errors 31.8 +/- 40. 5 vs. 19.2 +/- 26.7), p < 0.001]. CONCLUSIONS: The attention test can be helpful for the measurement of daytime sleepiness, and CPAP therapy can improve test performance.

Accidents, Traffic↗
PubMed1998

STAT 5 and NF-Y are involved in expression and growth hormone-mediated sexually dimorphic regulation of cytochrome P450 3A10/lithocholic acid 6beta-hydroxylase.

The level of expression of a number of sexually differentiated liver proteins is primarily determined by plasma growth hormone (GH). Adult males have a pulsatile profile of GH release, while females have a relatively steady-state pattern of GH release. An important subset of these sexually differentiated hepatic proteins is certain cytochrome P450s (P450s). CYP3A10/6beta-hydroxylase is a male-specific P450 that catalyzes 6beta-hydroxylation of lithocholic acid, and the pattern of GH secretion is directly responsible for male-specific expression of this gene. The DNA element involved in GH-mediated regulation of CYP3A10/6beta-hydroxylase promoter activity binds a member of the STAT (signal transducers and activators of transcription) family of proteins. In this study we functionally demonstrate that two members of the STAT family, STAT 5a and STAT 5b, mediate GH-dependent regulation of CYP3A10/6beta-hydroxylase promoter activity. Furthermore, a neighboring DNA element binds NF-Y, a transcription factor involved in maintaining high levels of transcription of many genes and known to functionally interact with other factors. In the CYP3A10/6beta-hydroxylase gene, NF-Y also modulates binding of STAT 5, thereby modulating GH-mediated activation of its transcription.

Animals↗
PubMed1995

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.

Amino Acid Sequence↗
PubMed1994

Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice.

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level.

Animals↗
PubMed1993

The lithocholic acid 6 beta-hydroxylase cytochrome P-450, CYP 3A10, is an active catalyst of steroid-hormone 6 beta-hydroxylation.

CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6 beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6 beta-hydroxylation corresponding to a major activity in all three instances. CYP 3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6 beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16 alpha-hydroxylation, progesterone 16 alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar Vmax. values for the 6 beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower Km (25 microM, as against 75 microM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates:steroid hormones and bile acids.

Androstenedione↗
PubMed1992

Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.

Amino Acid Sequence↗