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M J Haas

Publications and source records attributed to M J Haas.

At least 19 recordsLinked to original sources
PubMed2001

Isolation and identification of a degradation product in a capsule formulation containing the elastase inhibitor, DMP 777.

An unexpected degradation product, greater than 0.10%, was observed in a DMP 777 capsule formulation stored at 40 degrees C/75% r.h. for 3 months and 25 degrees C/60% r.h. for 2 years. The degradant of interest was prepared in quantity by refluxing the drug substance in dilute acid. A preparative HPLC method was developed to separate the various degradants and to collect each as a separate fraction. Each fraction was analyzed by the analytical HPLC gradient test method to assure positive identification of each peak and to correlate each peak to the original capsule sample. Key isolated degradation products were used for structure elucidation with mass spectrometry and NMR. The major degradant of interest in the capsule formulation was found to be a carboxylic acid resulting from the acid hydrolysis of an amide bond.

Acids↗
PubMed2001

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor.

Serum apolipoprotein A(1) (apoA(1)) concentration is inversely correlated with the risk of premature atherosclerosis. Serum apoA(1) concentrations are regulated, in part, at the transcriptional level. ApoA(1) mRNA is synthesized primarily in the liver and small intestine, under the direction of a number of signaling molecules and tissue-specific regulatory elements. Previously, we demonstrated that extracellular acidosis suppresses apoA(1) mRNA levels at the level of transcription. Here we demonstrate that intracellular acidosis, in the absence of extracellular pH changes, represses apoA(1) promoter activity. Repression occurs through a pH responsive element (pH-RE) located within the apoA(1) gene promoter. Acidosis increases the specific DNA binding activity of a putative repressor protein within the immediate 5'-flanking region of the apoA(1) gene. The cis-element that binds the putative repressor protein contains a negative thyroid hormone response element (nTRE) located 3' and adjacent to the apoA(1) TATA box. Mutation of the nTRE/pH-RE abrogates protein binding and alters the activity of reporter genes controlled by this element. Repression by acidosis did not require de novo mRNA and protein synthesis. Inhibition of tyrosine kinase activity and diacylglycerol-stimulated protein kinase C (PKC) signaling pathways with tyrophostin A47 and phorbol myristate acetate, respectively, did not affect the repression of apoA(1) promoter activity with acidosis. These results suggest that transcriptional repression of the apoA(1) gene by alterations in ambient pH is associated with enhanced DNA binding activity of a repressor protein, through a mechanism which appears to be independent of de novo mRNA and protein synthesis, tyrosine kinase activity, or PKC activation.

Acidosis↗
PubMed2000

Troglitazone stimulates acetyl-CoA carboxylase activity through a post-translational mechanism.

Troglitazone, a thiazolidinedione, is known to act as an insulin sensitizer. The various effects of the drug include stimulation of glucose utilization and inhibition of gluconeogenesis and fatty acid oxidation. We studied the effect of troglitazone treatment on rat liver acetyl-CoA carboxylase (ACC), the key enzyme that catalyzes the formation of malonyl-CoA, the rate-limiting step in the synthesis of long chain fatty acids. Treatment of rats with troglitazone for 18 days resulted in more than 200% increase in the activity of hepatic acetyl-CoA carboxylase (1.01+/-0.14 and 2.33+/-0.28 mU/mg supernatant protein for control and troglitazone-treated rats, respectively) (p<0.001). The expression of acetyl-CoA carboxylase mRNA, as studied by RNAse protection assay, was not significantly different between the two groups of animals. The ACC from control and troglitazone-treated groups was purified by avidin-affinity chromatography. The purified enzyme migrated as a major protein band (Mr 262,000) on SDS-polyacrylamide gels. Troglitazone treatment was associated with increased citrate sensitivity of ACC. The specific activity of the purified preparation in troglitazone-treated rats was increased by 67% (2.5 vs. 1.5 U/mg). Quantitation of alkali-labile phosphate content of the purified preparation revealed 5.66+/-0.17 and 6.29+/-0.13 mol Pi/mol subunit of 262 Kda for control and troglitazone-treated rats, respectively (P<0.01). The subtle increase in phosphate content does not explain the observed activation of the enzyme. It is possible that additional mechanisms such as troglitazone related rearrangement of the occupancy of select phosphate binding sites or altered binding of the biotin cofactor may also contribute to the observed activation of ACC.

Acetyl-CoA Carboxylase↗
PubMed2000

Sequence analysis of the human glycoprotein hormone alpha-subunit gene 5'-flanking DNA and identification of a potential regulatory element as an alu repetitive sequence.

The nucleotide sequence of the human glycoprotein hormone alpha-subunit (GPHalpha) gene 5'-flanking DNA was determined from -1637 to +49 relative to the cap site (+1). Comparison of the upstream sequence of the human gene with those of rhesus and mouse demonstrates regions with variable identity. When the 1.7 kb fragment was used to drive the expression of chloramphenicol acetyltransferase (CAT) in transiently transfected HeLa cells, it was found that CAT activity was elevated about 3-fold when the fragment was truncated from -1637 to -846, suggesting the presence of a negative regulatory element in the distal 5'-flanking DNA. This overlaps an Alu repetitive sequence (ARS) located between nucleotides -1330 and -1007. Gel mobility shift and DNase protection analyses identified a protein binding site centered around -1100 in the ARS second monomer. The GPHalpha upstream ARS was cloned in both orientations in positions upstream and downstream from the bacterial CAT gene under control of the herpes simplex virus thymidine kinase (tk) promoter. DNA-mediated transient transfection of these plasmids revealed a marked inhibition (79-82%) of CAT production by the ARS when it was cloned upstream from the tk promoter and in the same orientation as that found in the GPHalpha 5'-flanking DNA. Smaller decreases (29-57%) were produced by the ARS cloned upstream from the tk promoter in the reverse orientation. In marked contrast, the Alu repetitive element had little or no effect when cloned in either orientation downstream from the tk-CAT gene. Introduction of a second ARS downstream from the CAT reporter gene in vectors already containing an ARS upstream from the tk promoter significantly reduced the strong negative effect elicited by the upstream repetitive element. When compared to the Blur 8 Alu element, the GPHalpha upstream ARS differs markedly with respect to its effect on tk-CAT expression in transient assays and as a substrate for DNA binding proteins present in HeLa nuclear extracts. Together, the transient expression results demonstrate that ARS elements can influence expression of nearby class II promoters. The extent of this effect depends on element position and orientation, cell type, the particular ARS (e.g., GPHalpha or Blur 8), and whether copies were present both upstream and downstream from the transcription unit.

Alu Elements↗
PubMed2000

Monosaccharide-enriched diets cause hyperleptinemia without hypophagia.

To determine the effect of monosaccharide-enriched diets on plasma leptin and food consumption, body weight, food intake, and serum glucose, insulin, and leptin concentrations were measured in rats maintained on a 10-d course of 60% glucose or 60% fructose diet. The serum leptin concentration in rats fed a high-glucose diet (7.60 +/- 0.6 ng/mL) or a high-fructose diet (5.12 +/- 0.8 ng/mL) was significantly increased compared with that in control rats (2.45 +/- 0.10 ng/mL; P < 0.001). To ascertain that the observed effect was related to hyperinsulinemia, a group of rats were infused with exogenous insulin or rendered insulin resistent with a high-fat diet. When hyperinsulinemia was induced with exogenous infusion, the serum leptin was increased (5.56 +/- 0.23 ng/mL; P < 0.001). High-fat feeding was associated with increased serum leptin (12.1 +/- 1.4 ng/mL) and insulin levels. The increased serum leptin concentration was not associated with decreased food intake. We conclude that monosaccharide-enriched diets, probably through hyperinsulinemia or relative or absolute insulin resistance, cause hyperleptinemia, which does not appear to change feeding behavior.

Animals↗
PubMed2000

Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography.

A novel assay for measuring the free leptin fraction was developed and validated against a chromatographic technique. The assay used acid-ethanol extraction (AEE) for separation of bound/free leptin moieties. The interassay coefficient of variation was 3.9%. The specificity for leptin binding was confirmed by incubation with 1 microg of unlabeled rat leptin that effectively competed with radiolabeled leptin whereas human growth hormone and interleukin-6 were ineffective in competing with radiolabeled leptin binding. Scatchard analysis of competitive binding experiments with rat plasma demonstrated a linear relationship with a binding affinity of 0.3-0.6 x 109 M-1. This novel assay was used to determine if age-related insensitivity to leptin action is secondary to altered serum leptin binding. Rats at various age groups were studied for changes in body adiposity and serum total and free leptin concentrations. Serum free leptin concentrations (ng/ml mean +/- SEM) were significantly increased in 24-month-old rats (5.56 +/- 0. 21) compared with 18-month-old rats (4.76 +/- 0.17) (P < 0.01) despite similar body weight and adiposity of the two age groups. The increase in plasma free leptin concentrations in 12-month-old rats (3.86 +/- 0.28) and 6-month-old rats (2.05 +/- 0.06) relative to 3-month-old rats (1.37 +/- 0.06) (P < 0.001) was out of proportion to the increase in body adiposity in aging rats. It is concluded that aging in rats is associated with relative insensitivity to leptin. This change cannot be attributed to increased plasma binding or to a reduction in the leptin free fraction.

Adipose Tissue↗
PubMed2000

Age-related changes in rat hepatic acetyl-coenzyme A carboxylase.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in the synthesis of long-chain fatty acids. Since aging influences adiposity, we studied the activity of ACC and its mRNA content in livers of 4-, 12-, and 24-month-old male Fischer 344 rats. The mean (+/- SEM) activity of ACC (mU/mg protein) in liver homogenates from 4-month-old rats was 1.01 +/- 0.14. There was an 80% increase in activity (1.83 +/- 0.27) in 12-month-old rats (P < 0.01). However, there was significantly less activity (0.46 +/- 0.06) in livers of 24-month-old rats (P < 0.001). The total activity of ACC (per g liver) followed the same trend. The enzyme from all age groups was purified by avidin-affinity chromatography. The purified preparation migrated as a major protein band (M(r) 262,000) on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The specific activity of the purified preparation was 1.5, 1.8, and 1.8 U/mg for 4-, 12-, and 24-month-old rats, respectively. The alkali-labile phosphate content was 5.66 +/- 0.17, 5.64 +/- 0.21, and 6.21 +/- 0.35 mols P(i)/mole subunit for 4-, 12-, and 24-month-old rats, respectively. These age-related differences were not significant. The hepatic ACC mRNA measured by ribonuclease protection assay when corrected for G3PDH mRNA was significantly reduced in 24-month-old rats (0.24 +/- 0.03) compared with 12-month-old (0.58 +/- 0.04) or 4-month-old rats (0.43 +/- 0.007) P < 0.01. In summary: (i) Aging in rats is associated with significant changes in ACC activity; (ii) the purified ACC preparations from the three age groups had similar specific activity and similar phosphate content; and (iii) the changes in ACC mRNA content of the liver paralleled the changes in total enzyme activity when 12-month-old rats were compared with 24-month-old rats whereas the increase in ACC activity in 12-month-old rats compared with 4-month-old rats could not be ascribed to changes in hepatic mRNA levels. These results indicate that the age-related changes in hepatic ACC occur at a post-translational level during early years of aging and at a pretranslational level at late states of senescence. These changes may contribute to the age-related alterations in body adiposity.

Acetyl-CoA Carboxylase↗
PubMed2000

Induction of the apolipoprotein AI gene by fasting: a relationship with ketosis but not with ketone bodies.

Apolipoprotein AI (apoAI) expression is inversely related to the incidence of atherosclerosis. ApoAI expression is also influenced by the nutritional state and diabetes. We used both cell culture and animal models to examine the effect of fasting and ketoacidosis on apoAI gene expression. Two days of food deprivation in rats increased hepatic and intestinal apoAI mRNA by 2.6- and 2.3-fold, respectively (P < .05). The absolute concentration of plasma apoAI did not change. However, the plasma apoAI concentration relative to the plasma concentration of serum proteins was increased 23% (P < .05). In fasting rats, there was a significant positive correlation between the serum beta-hydroxybutyrate concentration and hepatic or intestinal apoAI mRNA level. Despite this correlation, changes in apoAI mRNA are probably not mediated by ketone bodies, since neither hepatic nor intestinal apoAI mRNA levels were altered in rats maintained on a ketogenic diet for 10 days or treated with isobutyramide, an orally active ketone analog. In addition, the activity of the rat apoAI promoter was not altered in Hep G2 cells treated with isobutyramide or fatty acids or exposed to hypoglycemic conditions, while dexamethasone increased promoter activity 1.9-fold (P < .05). These data indicate that metabolic changes other than ketone bodies, such as an increase in plasma glucocorticoids, may account for starvation-induced expression of apoAI.

3-Hydroxybutyric Acid↗
PubMed2000

Performance of a group B streptococcal prophylaxis protocol combining high-risk treatment and low-risk screening.

OBJECTIVE: This study was undertaken to evaluate a group B streptococcal protocol in a large community hospital that combined treatment of high-risk patients with rapid screening of low-risk patients. STUDY DESIGN: In a prospective cohort study from 1994 through 1996 laboring patients in a level III community hospital were considered to be at high risk for neonatal group B streptococcal transmission if they were at <37 weeks' gestation, if they had rupture of membranes >12 hours, if they were known carriers of group B streptococci, if they had a temperature > or =100 degrees F, if the gestation was complicated by fetal growth restriction or was a multiple gestation, or if they had a previous neonate infected with group B streptococci. High-risk patients were treated intravenously with antibiotics during labor. Low-risk patients were screened for group B streptococcal antigen by means of a rapid optical immunoassay. Patients with positive screening results were treated. Neonatal morbidity and mortality were evaluated. RESULTS: Two of 9932 infants delivered had group B streptococcal sepsis diagnosed. In the 2 previous years without a protocol 9 cases of neonatal group B streptococcal sepsis had been diagnosed in 8188 deliveries (P =.0287 by Fisher exact test). The 2 cases of group B streptococcal sepsis during the protocol were as follows: 1 infant born to a high-risk mother with delay in treatment and 1 infant born to a low-risk mother with negative results of both culture and rapid screen during labor. During the previous period 7 infected infants had been born to high-risk mothers and 2 had been born to low-risk mothers. The maternal group B streptococcal carriage rate during the study was 18%. Group B streptococcal rapid optical immunoassay sensitivity was 81%. Elapsed time from screening to treatment was < or =2(1/2) hours for 93% of patients. No maternal anaphylaxis, no increase in bacterial neonatal sepsis caused by organisms other than group B streptococci, and no protocol-related group B streptococcal antibiotic resistance were noted. CONCLUSION: Successful implementation and maintenance of a protocol combining treatment of high-risk patients with rapid screening of low-risk patients during labor reduced neonatal group B streptococcal sepsis.

Adult↗
PubMed2000

Multiple polypeptide hormone expression in pancreatic islet cell carcinomas derived from phosphoenolpyruvatecarboxykinase-SV40 T antigen transgenic rats.

Transgenic rats carrying a PEPCK-SV40 large T-antigen (TAg) transgene rapidly develop numerous pancreatic islet cell neoplasms, the cells of which express TAg. Although many of the larger neoplasms contain relatively undifferentiated cells, many tumors contain areas of well-differentiated cells with abundant endoplasmic reticulum (ER) and secretory granules for endocrine hormones like those observed in normal pancreatic islets. In the well-differentiated lesions, glucagon-producing alpha-cells, insulin-producing beta-cells, and somatostatin-producing delta-cells are readily identifiable morphologically under the electron microscope. Beta-cells were observed in all normal and hyperplastic islets, and nests of these cells were scattered throughout the larger neoplasms. These nests varied from small clusters of epithelium-like cells that stain intensely for insulin, to sheets of small, basophilic cells that stain more diffusely for the hormone. Alpha-cells were also present in all of the normal and hyperplastic islets, but in larger hyperplastic islets, the peripheral localization was absent. Larger neoplasms contained many nests of glucagon-expressing cells, as well as scattered glucagon-producing single cells. Delta-cells were rarely observed in the hyperplastic islets and in the neoplasms. Blood-glucose levels were unaltered in the transgenic animals relative to their nontransgenic litter mates. Thus although these islet cell neoplasms express several polypeptide hormones, there is no obvious clinical effect of such expression in vivo.

Animals↗
PubMed2000

Effects of ketoacidosis on rat apolipoprotein A1 gene expression: a link with acidosis but not with ketones.

To determine if ketoacidosis contributes to reduced apolipoprotein A1 (apoA1) expression in insulin-deficient diabetic rats, we examined the regulation of apoA1 gene expression in response to changes in ambient pH or ketone body concentrations. Hepatic apoAI mRNA levels were reduced 42% in diabetic rats relative to nondiabetic controls (means+/-s.d.; 321.8+/-43.7 vs 438.7+/-58.8 arbitrary units; P<0.03). Neither endogenous apoA1 mRNA nor transcriptional activity of the rat apoA1 gene promoter (from -474 to -7) were altered by sodium butyrate or isobutyramide (0.3 mM to 10 mM) in Hep G2 or Caco-2 cells. Rat hepatic and intestinal apoA1 mRNA levels, and plasma apoA1 concentration, were not altered 24 h after isobutyramide administration (500 mg/kg by gavage). When the effect of altering ambient pH within a wide range commonly encountered in vivo was studied, acidosis (pH 6.7), relative to alkalosis (pH 7.9), decreased apoAI mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA by 47% in Hep G2 cells (P<0.025) and by 24% in Caco-2 cells (P<0.017). Acidosis did not alter cytomegalo virus (CMV)-beta-galactosidase activity, or the activity of the simian virus (SV40) early-region promoter, in either cell line transfected with the respective constructs. The lowering of ambient pH was associated with a graded reduction in apoAI promoter activity. At pH 6.7, apoAI promoter activity was reduced by 75% compared with promoter activity at pH 7.9. These observations indicate that acidosis, but not ketosis, contributes to the reduction in apoA1 expression during diabetic ketoacidosis by down-regulating apoAI promoter activity.

Amides↗
PubMed2000

Lipase-catalysed production of biodiesel fuel from some Nigerian lauric oils.

Fatty acids esters were produced from two Nigerian lauric oils, palm kernel oil and coconut oil, by transesterification of the oils with different alcohols using PS30 lipase as a catalyst. In the conversion of palm kernel oil to alkyl esters (biodiesel), ethanol gave the highest conversion of 72%, t-butanol 62%, 1-butanol 42%, n-propanol 42% and iso-propanol 24%, while only 15% methyl ester was observed with methanol. With coconut oil, 1-butanol and iso-butanol achieved 40% conversion, 1-propanol 16% and ethanol 35%, while only traces of methyl esters were observed using methanol. Studies on some fuel properties of palm kernel oil and its biodiesel showed that palm kernel oil had a viscosity of 32.40 mm2/s, a cloud point of 28 degrees C and a pour point of 22 degrees C, while its biodiesel fuel had a viscosity of 9.33 mm2/s, a cloud point of 12 degrees C and a pour point of 8 degrees C. Coconut oil had a viscosity of 28.58 mm(2)/s, a cloud point of 27 degrees C and a pour point of 20 degrees C, while its biodiesel fuel had a viscosity of 7.34 mm2/s, a cloud point of 5 degrees C and a pour point of -8 degrees C. Some of the fuel properties compared favourably with international biodiesel specifications.

Alcohols↗
PubMed1999

Sodium butyrate-mediated induction of the glycoprotein hormone alpha-subunit gene: requirement for continued protein synthesis, identification of a butyrate-responsive element, and inhibition of promoter activation by 2-deoxyglucose.

Production of the glycoprotein hormone alpha-subunit (GPH alpha) was enhanced by sodium butyrate (Btr) in HeLa cells. Induction of the HeLa alpha-subunit gene by Btr was inhibited by the simultaneous addition of cycloheximide (CHX), indicating a requirement for continued protein synthesis. Transient expression assays using plasmids containing the GPH alpha gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the GPH alpha promoter is inducible by Btr in HeLa cells, and this induction could be prevented by 2-deoxyglucose (dGlc). CAT production driven by the SV-40 early promoter, the cytochrome P-450-IA1 promoter, and the Rous sarcoma virus long terminal repeat was also enhanced by Btr, but the augmented synthesis was not inhibited by the addition of dGlc, demonstrating that the effect is restricted only to some promoters. CAT synthesis could be induced by Btr when the GPH alpha promoter extended upstream to position -169 (relative to the transcription start site at +1) but not when the promoter terminated at -150, classifying the DNA between these termini as a Btr-responsive element (BRE). This region overlaps the composite trophoblast-specific enhancer. Inactivation of enhancer subdomains by site-directed mutagenesis confirmed the deletion analysis and ranked their response to Btr as CRE < TSE < URE < alpha ACT. Electrophoretic mobility shift analysis failed to detect any significant difference among several enhancer binding proteins in nuclear extracts from untreated and Btr-treated cells. Together, these results suggest that Btr-mediated induction of the alpha-subunit gene in HeLa cells is manifest either through the synthesis of a new transcription factor(s), which is inhibited by CHX but required for increased transcription from the GPH alpha gene promoter, or through the activity of existing factors that may require glycosylation or phosphorylation by a modification system that is inducible by Btr and inhibited by dGlc and CHX. These results further suggest that the factor is not an enhancer-binding protein or that Btr increases its transactivation potential without altering its DNA-binding activity.

Base Sequence↗
PubMed1999

Glucocorticoids stimulate CREB binding to a cyclic-AMP response element in the rat serine dehydratase gene.

Transcription of the rat serine dehydratase (SDH) gene, which is stimulated in hepatocytes by glucagon through the activity of the second messenger, cAMP, is augmented by pretreatment with glucocorticoids. A putative cAMP response element (CRE) located approximately 3.5 kbp upstream of the transcriptional start site was hypothesized to be responsible for this effect. Here we have demonstrated by DNaseI footprinting and site-directed mutagenesis that the phosphorylated cAMP response element binding protein (CREB) binds to a cAMP response element different from that described previously. While the amount of CREB in the extracts is unaltered by hormone treatment, more CREB is capable of binding the response element upon addition of dexamethasone (Dex). These studies suggest that synergistic induction of the SDH gene by cAMP and Dex is through a CRE and is due, in part, to regulation of CREB-DNA binding by treatment of the cells with glucocorticoids.

Animals↗
PubMed1999

Lipase-catalyzed fractionation of conjugated linoleic acid isomers.

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.

Chemical Fractionation↗
PubMed1999

Transgene expression and repression in transgenic rats bearing the phosphoenolpyruvate carboxykinase-simian virus 40 T antigen or the phosphoenolpyruvate carboxykinase-transforming growth factor-alpha constructs.

Transgenic Sprague-Dawley rats expressing either human transforming growth factor-alpha (TGFalpha) or simian virus 40 large and small T antigen (TAg), each under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, were developed as an approach to the study of the promotion of hepatocarcinogenesis in the presence of a transgene regulatable by diet and/or hormones. Five lines of PEPCK-TGFalpha transgenic rats were established, each genetic line containing from one to several copies of the transgene per haploid genome. Two PEPCK-TAg transgenic founder rats were obtained, each with multiple copies of the transgene. Expression of the transgene was undetectable in the TGFalpha transgenic rats and could not be induced when the animals were placed on a high-protein, low-carbohydrate diet. The transgene was found to be highly methylated in all of these lines. No pathological alterations in the liver and intestine were observed at any time (up to 2 years) during the lives of these rats. One line of transgenic rats expressing the PEPCK-TAg transgene developed pancreatic islet cell hyperplasias and carcinomas, with few normal islets evident in the pancreas. This transgene is integrated as a hypomethylated tandem array of 10 to 12 copies on chromosome 8q11. Expression of large T antigen is highest in pancreatic neoplasms, but is also detectable in the normal brain, kidney, and liver. Mortality is most rapid in males, starting at 5 months of age and reaching 100% by 8 months. Morphologically, islet cell differentiation in the tumors ranges from poor to well differentiated, with regions of necrosis and fibrosis. Spontaneous metastasis of TAg-positive tumor cells to regional lymph nodes was observed. These studies indicate the importance of DNA methylation in the repression of specific transgenes in the rat. However, the expression of the PEPCK-TAg induces neoplastic transformation in islet cells, probably late in neuroendocrine cell differentiation. T antigen expression during neoplastic development may result in a pervasive change in the islet cell growth properties with selection of a transformed phenotype as a possible requirement for cell viability.

Animals↗
PubMed1998

Isolation of an Alu repetitive DNA binding protein and effect of CpG methylation on binding to its recognition sequence.

The structure, expression, and evolution of Alu repetitive DNA elements have been extensively studied, but the role of these sequences in the function of primate genomes has yet to be elucidated. The contribution of Alu repetitive sequences (ARS) to the structure, maintenance, or expression of the human genome is undoubtedly mediated by one or more DNA binding proteins. As part of a larger study in this laboratory to define the molecular mechanisms that result in de-repression of the glycoprotein hormone alpha-subunit (GPH alpha) gene in a variety of tumor cell types, it was found that the gene was hypermethylated in a variety of cell lines that produce alpha-subunit at high levels and significantly less methylated in cell lines where the gene is unexpressed or expressed at low levels. This is in sharp contrast to the majority of genes examined in this regard, which show an inverse correlation between methylation and expression. The analysis was extended to a group of clones isolated from a single cell line (HeLa) that were differentially methylated over the GPH alpha gene and exhibited a 400-fold range in its expression. These analyses demonstrated that methylation of a small number of CpG dinucleotides correlated with high level expression of the gene. Two of these sites are imbedded in oppositely oriented Alu repeats located in the 5'-flanking DNA and second intron. The upstream site was examined in some detail. DNase I footprint analysis demonstrated that the protein protects a region encompassing the sequence 5'-TTGAACCCGGGAG-3', and electrophoretic gel mobility shift analysis demonstrated specific binding of a protein to an oligonucleotide containing the DNase footprint sequence. Chromatography of nuclear extracts on Sephacryl S-200, heparin--agarose, and oligonucleotide--Sepharose produced an apparently homogeneous preparation of the 50-53 kDa DNA-binding protein as judged by silver staining of sodium dodecylsulfate polyacrylamide gels. The affinity-purified material was enriched 15- to 18,000-fold over crude nuclear extracts. Binding of this protein to an oligonucleotide containing the DNase-protected sequence was severely inhibited when CpG dinucleotide in the Msp I recognition site was methylated on either the sense or antisense strands. Based on its properties, this protein has been termed MeSABp50 for methylation-sensitive Alu binding protein of 50 kDa.

Base Sequence↗