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PubMed1984

Activation of immunoglobulin mu gene expression involves stepwise demethylation.

In an attempt to identify stages of mu gene activation subsequent to VHDHJH assembly, we investigated two pre-B cell lines (A1 and A8) that have both alleles of the JH locus rearranged but do not make mu polypeptides. The block in A1 reflects incorrect VHDHJH assembly on both alleles. In A8, although an out-of-phase VHDHJH-C mu allele is transcribed, the properly assembled allele is silent, despite having a normal VH promoter and mu enhancer. Thus, transcription can be restricted to a single mu allele. Low level mu transcription in both lines was associated with demethylation of the VHDHJH and enhancer regions but not of the C mu gene. Markedly elevated mu transcription ensued on lipopolysaccharide stimulation or fusion to a plasmacytoma, but only fusion induced C mu demethylation. Hence stepwise demethylation is implicated in mu gene regulation, but enhanced expression can also occur independently of, or prior to, demethylation.

Alleles↗
PubMed1986

Plasma levels of the prodrug, arbaprostil, [(15R)-15-methylprostaglandin E2], and its active, antiulcer (15S) epimer in humans after single dose oral administration.

Arbaprostil [(15R)-15-methylprostaglandin E2] is an antiulcer prodrug being evaluated for the treatment of gastric and duodenal ulcers in humans. It epimerizes in acidic gastric fluid to produce the biologically active form, (15S)-15-methyl-PGE2, which acts directly on the gastric mucosa and possesses both gastric acid antisecretory and cytoprotective properties. Because of its local mode of action, plasma levels of the two epimers may have greater relevance to drug safety than to therapeutic efficacy. In the present study, plasma concentrations of both 15-methyl-PGE2 epimers resulting from a gastric acid antisecretory dose of arbaprostil oral solution (50 micrograms) were measured in eight male volunteers having sufficient gastric acidity for prodrug activation (pH less than 3). Arbaprostil was determined with a newly developed RIA having a sensitivity of 10 pg X mL-1. The accuracy of the RIA was confirmed by parallel analysis of plasma samples by HPLC. (15S)-15-Methyl-PGE2 was also determined by HPLC. Arbaprostil was both rapidly absorbed and eliminated (tmax of 15-30 min and plasma t1/2 of 20 min), but there was large intersubject variability in its observed maximum plasma concentration (38 to 348 pg X mL-1). The concentration of (15S)-15-methyl-PGE2 did not exceed 25 pg X mL-1 In six subjects and 50 pg X mL-1 in the remaining two subjects. The significance of these results is discussed.

Administration, Oral↗
PubMed2005

p16 expression in Barrett's esophagus and esophageal adenocarcinoma: association with genetic and epigenetic alterations.

Alteration of the p16 tumor suppressor gene has been implicated as a critical lesion in the molecular pathogenesis of esophageal adenocarcinoma. The aim of this study was to characterize the spectrum of p16 alterations in surgically resected esophageal tissues, comprising histologically normal esophageal squamous and gastric epithelia, premalignant Barrett's epithelia, and associated esophageal adenocarcinomas, and to explore associations between p16 mRNA expression and p16 mutations, deletions, promoter hypermethylation, p16 protein expression, and clinico-pathologic features for the same tissues. We have shown that while p16 mutations are uncommon (2%; 1/54), hypermethylation of the p16 promoter is detected in 43% (9/21) of histologically normal epithelia, in 77% (14/18) of associated Barrett's epithelia, and in 85% (18/21) of esophageal adenocarcinomas. However, p16 mRNA levels (relative to matched normal epithelia) were variable in Barrett's epithelia and adenocarcinomas, having no clear correlation with methylation status or other molecular and clinico-pathological parameters. These findings are consistent with a role for the p16 tumor suppressor gene early in the molecular progression of Barrett's epithelium to invasive esophageal adenocarcinoma, but do not support the notion that the detection of hypermethylation is systematically associated with low levels of expression.

Adenocarcinoma↗
PubMed1995

Effects of L-arginine and L-nitro-arginine methyl ester on recovery of neonatal lamb hearts after cold ischemia. Evidence for an important role of endothelial production of nitric oxide.

Myocardial ischemia and reperfusion results in both ventricular and endothelial dysfunction. We have found that the endothelial defect is a reduced vasodilator response to an intraarterial infusion of acetylcholine that is likely due to reduced nitric oxide release, and we have hypothesized that reduced endothelial nitric oxide production contributes to postischemic cardiac dysfunction. However, others report that nitric oxide is deleterious after ischemia. We therefore examined the effects of infusions of L-arginine (3 mmol/L), a precursor of nitric oxide, D-arginine (3 mmol/L), an inactive stereoisomer of L-arginine, L-nitro-arginine methyl ester (1 mmol/L); a competitive inhibitor of nitric oxide synthase, and L-nitro-arginine methyl ester (1 mmol/L) plus L-arginine (3 mmol/L) versus controls in isolated blood-perfused neonatal lamb hearts having 2 hours of cold cardioplegic ischemia. L-nitro-arginine methyl ester was given before reperfusion, and L-arginine and D-arginine were infused for the first 20 minutes of postischemic reperfusion. At 30 minutes of reperfusion, by comparison with the control group, the L-arginine group showed significantly better recovery (p < 0.05) of left ventricular systolic function (maximum developed pressure, developed pressure at V10 [balloon volume to produce an end-diastolic pressure of 10 mm Hg during baseline measurement], positive maximum dP/dt, and dP/dt at V10), diastolic function (negative maximum dP/dt), coronary blood flow, and endothelial function assessed by the coronary vascular resistance response to acetylcholine. The L-nitro-arginine methyl ester hearts showed a significantly poorer recovery (p < 0.05) in left ventricular function, coronary blood flow, and endothelial function than the control group. These effects of L-nitro-arginine methyl ester were reversed to equal control values by adding a 3 mmol/L concentration of L-arginine to L-nitro-arginine methyl ester. There were no significant differences in the recovery of any variables between the D-arginine and control groups. These results point to an important salutary role for the endothelial production of nitric oxide in cardiac recovery after hypothermic ischemia in neonatal lamb hearts. The mechanism of these beneficial effects of L-arginine after ischemia and reperfusion is likely due to enhancement of the endothelial production of nitric oxide.

Acetylcholine↗
PubMed1991

New isomeric classes of topically active ocular hypotensive carbonic anhydrase inhibitors: 5-substituted thieno[2,3-b]thiophene-2-sulfonamides and 5-substituted thieno[3,2-b]thiophene-2-sulfonamides.

A series of 5-substituted thieno[2,3-b]- and thieno[3,2-b)- and thieno[3,2-b)thiophene-2-sulfonamides was prepared and evaluated for topical ocular hypotensive activity in glaucoma models. The 5-substituents were varied to maximize both inhibitory potency against carbonic anhydrase and water solubility. At the same time, these substituents were varied in order to obtain compounds with the appropriate pKa to minimize pigment binding in the iris. All of these variables were optimized in the best compound, 5-[[(methoxyethyl)[(methoxyethyl)ethyl] amino]methyl]thieno[2,3-b]thiophene-2-sulfonamide hydrochloride (55).

Animals↗
PubMed1998

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells.

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.

Animals↗
PubMed1991

Relationships between the proton nuclear magnetic resonance properties of plasma lipoproteins and cancer.

We conducted a comprehensive investigation of the origin of nuclear magnetic resonance (NMR) lineshape variability of plasma lipids among healthy individuals and those with cancer. The methyl and methylene resonances of lipid in human plasma, whose linewidths have been reported to correlate with the presence of malignancy, are composed of the overlapping resonances of "mobile" protons from the major lipoproteins (very-low-, low-, and high-density lipoproteins). We tested two hypotheses for the origin of the narrower plasma linewidths observed for cancer patients: (a) malignancy-associated differences in the spectral properties (chemical shift, lineshape) of one or more of the lipoproteins, and (b) differences in the fraction of lipoprotein lipid giving rise to detectable NMR signal. Analysis of the concentrations of lipoprotein lipid and of 500 MHz NMR spectra of the lipoprotein constituents in greater than 100 plasma samples failed to provide support for either hypothesis. Although linewidths were found to be significantly narrower for the cancer group, the difference is entirely attributable to differences in the concentrations of the lipoproteins.

Adult↗
PubMed1997

Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with a characteristic pattern of visceromegaly and predisposition to childhood tumours. BWS is a genetically heterogeneous disorder; most cases are sporadic but approximately 15% are familial and a small number of BWS patients have cytogenetic abnormalities involving chromosome 11p15. Genomic imprinting effects have been implicated in familial and non-familial BWS. We have investigated the molecular pathology of 106 sporadic BWS cases; 17% (14/83) of informative cases had uniparental disomy (UPD) for chromosome 11p15.5. In each case UPD appeared to result from a postzygotic event resulting in mosaicism for segmental paternal isodisomy. The critical region for isodisomy was refined to a 25 cM interval between D11S861 and D11S2071 which contained the IGF2, H19, and p57(KIP2) genes. In three cases isodisomy for 11q markers was detected but this did not extend further than 11q13-q21 suggesting that complete chromosome 11 disomy may not produce a BWS phenotype. The allele specific methylation status of the H19 gene was investigated in 80 sporadic BWS cases. All 13 cases with UPD tested displayed hypermethylation consistent with an excess of paternal H19 alleles. In addition, five of 63 (8%) cases with normal biparental inheritance had H19 hypermethylation consistent with an "imprinting centre" mutation (ICM) or "imprinting error" (IE) lesion. The phenotype of patients with putative ICM/IE mutations was variable and overlapped with that of non-UPD sporadic BWS cases with normal H19 methylation. However, exomphalos was significantly (p < 0.05) more common in the latter group. These findings may indicate differential effects on the expression of imprinted genes in chromosome 11p15 according to the precise molecular pathology. Analysis of H19 methylation is useful for the diagnosis of both UPD or altered imprinting in BWS and shows that a variety of molecular mechanisms may cause relaxation of IGF2 imprinting in BWS.

Aneuploidy↗
PubMed1993

Developing a stable bilateral model of parkinsonism in rhesus monkeys.

The non-human primate models of Parkinson's disease which have been developed using the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine) have proven to be either unstable or variable, or to display only a limited subset of parkinsonian features. The present study examined a new two-stage lesion approach in which MPTP was administered via the carotid arteries. The first infusion through one artery produced a hemiparkinsonian state and was followed several months later by a second MPTP infusion into the contralateral carotid artery to induce bilateral parkinsonism. Animals receiving lesions were evaluated using a battery of tests which included a monkey parkinsonism rating scale, a movement time-task and continuous monitoring of home cage activity. All animals monitored showed significant decreases in activity levels of up to 95% following the second lesion. These decreased activity levels remained stable throughout the observation period of up to 12 months postlesion. In addition to the decreased home cage activity, bilaterally lesioned animals displayed bilateral parkinsonian features including akinesia, bradykinesia, rigidity, tremor and balance and gait disturbances which were stable, following an acute period of up to 45 days, for the remainder of the study. Administration of levodopa increased activity levels and reduced motor dysfunctions. Thus, a two-stage bilateral lesion approach, utilizing the neurotoxin MPTP, appears to provide a less variable and relatively stable model of bilateral Parkinson's disease in nonhuman primates. Treated animals display the cardinal features of parkinsonism and respond appropriately to the standard antiparkinsonian drug, levodopa.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1993

Methyl jasmonate-regulated translation of nuclear-encoded chloroplast proteins in barley (Hordeum vulgare L. cv. salome).

The naturally occurring plant growth regulator (-)-jasmonic acid methyl ester (JaMe) induces the formation of novel abundant proteins in excised barley leaf segments. Concomitantly, this substance depresses the translation of most preexisting ("control") leaf mRNAs, including those for nuclear-encoded chloroplast proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU, rbcS gene product) and several light harvesting chlorophyll protein complex apoproteins (LHCPs, cab gene products). The changes in protein synthesis observed for SSU and LHCPs did not correspond to equivalent alterations in the rbcS and cab transcript levels. Analysis of polysome-associated in vitro translatable and hybridizable mRNAs, however, demonstrated a restriction of rbcS and cab transcripts to smaller polysomes in JaMe-exposed leaf tissues, in comparison to water-treated tissues. Since treatment of JaMe-incubated leaf segments with cycloheximide prior to harvest led to a shift of both transcripts toward larger polysomes, a hormone-induced impairment of chain initiation is assumed to lower translation of SSU and LHCP in situ. In contrast, the mRNA for plastid leucyl-tRNA synthetase (LRS1, lrs1 gene product) neither changed its abundance nor its association with polysomes in JaMe-treated leaves and was translated into the corresponding polypeptide. Together, our results highlight a remarkable variability of nuclear gene expression in response to plant growth regulators of the methyl jasmonate type.

Acetates↗
PubMed1999

Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha.

We examined cytokine-mediated neuronal death in neuron-astrocyte cultures from second trimester human fetal cerebrum. In these cultures, high-output inducible nitric oxide synthase (NOS) and tumor necrosis factor-alpha (TNFalpha) are expressed in astrocytes after exposure to IL-1beta/IFNgamma. Neuronal cell death was evident at >/=48 h following cytokine stimulation. Neutralizing anti-TNFalpha antiserum inhibited ( approximately 48%) neurotoxicity in IL-1beta/IFNgamma-treated cultures, demonstrating a role for endogenously produced TNFalpha. Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N-methyl D-aspartate (NMDA) receptor antagonists in these cultures was smaller and variable. Similarly, the effect of the NOS inhibitor, N(G)-monomethyl L-arginine (NMMA) on IL-1beta/IFNgamma-induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged. Neurons die by apoptosis in cytokine-treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL. Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL-1beta/IFNgamma in primary human neuron-astrocyte cultures and a crucial role for endogenous TNFalpha in mediating neurotoxicity in this system. These results firmly establish the neurotoxic potential of the inflammatory cytokines IL-1beta and TNFalpha in the human CNS.

Apoptosis↗
PubMed1996

NG methyl-L-arginine increases the hyperthermic effects of prostaglandin E1.

The sympathetic firing rate of the nerves innervating interscapular brown adipose tissue (IBAT), IBAT and colonic temperatures (TIBAT and TC) were monitored in urethane-anaesthetized male Sprague-Dawley rats. These variables were measured for a period of 40 min before (baseline values) and 40 min after a 2 mg NG-methyl-L-arginine (NMA) injection plus an intracerebroventricular administration of 500 ng prostaglandin E1 (PGE1) into a lateral cerebral ventricle. No drug was injected in control rats. The results show that NMA enhances the increases in firing rate, TIBAT and TC induced by PGE1. These findings indicate that an inhibitor of nitric oxide synthesis, such as NMA, increases the sympathetic and thermogenic responses to injection of PGE1.

Adipose Tissue, Brown↗
PubMed2006

Regulation of high molecular weight-melanoma associated antigen (HMW-MAA) gene expression by promoter DNA methylation in human melanoma cells.

The human high molecular weight-melanoma associated antigen (HMW-MAA) is a membrane-bound chondroitin sulfate proteoglycan that is variably expressed in a high percentage of melanoma cell lines and tumors. Since the mechanism(s) regulating HMW-MAA expression has(ve) not been defined, in this study, we have examined whether promoter DNA methylation regulates the level of HMW-MAA expression. In melanoma cell lines, the level of HMW-MAA mRNA and protein expression is coordinately regulated, implicating a transcriptional control mechanism. Consistent with a role for regulation by DNA methylation, we have found that a dense CpG island flanks the human HMW-MAA gene transcriptional start site. Methylation-specific PCR and sodium bisulfite DNA sequencing analyses indicate that the HMW-MAA promoter is heavily methylated in melanoma cell lines, melanoma lesions and normal lymphocytes that do not express HMW-MAA; in contrast, the HMW-MAA promoter is not methylated in melanoma cell lines and tumors that express this antigen. In addition, HMW-MAA expression is markedly induced in HMW-MAA-negative melanoma cell lines by incubation with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. In summary, our results establish DNA methylation as a key regulator of HMW-MAA expression by human melanoma cells. This information represents a useful background to optimize immunotherapeutic strategies targeting HMW-MAA.

Antigens, Neoplasm↗
PubMed1995

Variability of clinical and immunological phenotype in immunodeficiency-centromeric instability-facial anomalies syndrome. Report of two new patients and review of the literature.

Immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome is a condition characterized by variable combined immunodeficiency, developmental delay, facial anomalies and a variety of structural chromosomal rearrangements. Recently, aberrations at the molecular level have been described consisting of alterations in the methylation pattern of classical satellite DNA. To our knowledge 15 subjects have been described so far in the literature showing marked phenotypic variability. We report on two new patients with normal development and some peculiar clinical and immunological manifestations. All patients previously reported in the literature are reviewed. CONCLUSION. The identification of these two cases among our hypogammaglobulinaemic patients suggests that ICF syndrome is not a rare disorder and it should be always taken into account in immunodeficient patients with facial abnormalities.

Abnormalities, Multiple↗
PubMed2004

Classification of individual lung cancer cell lines based on DNA methylation markers: use of linear discriminant analysis and artificial neural networks.

The classification of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) can pose diagnostic problems due to inter-observer variability and other limitations of histopathology. There is an interest in developing classificatory models of lung neoplasms based on the analysis of multivariate molecular data with statistical methods and/or neural networks. DNA methylation levels at 20 loci were measured in 41 SCLC and 46 NSCLC cell lines with the quantitative real-time PCR method MethyLight. The data were analyzed with artificial neural networks (ANN) and linear discriminant analysis (LDA) to classify the cell lines into SCLC or into NSCLC. Models used either data from all 20 loci, or from five significant DNA methylation loci that were selected by a step-wise back-propagation procedure (PTGS2, CALCA, MTHFR, ESR1, and CDKN2A). The data were sorted randomly by cell line into 10 different data sets, each with training and testing subsets composed of 71 and 16 of the cases, respectively. Ten ANN models were trained using the 10 data sets: five using 20 variables, and five using the five variables selected by step-wise back-propagation. The ANN models with 20 input variables correctly classified 100% of the cell lines, while the models with only five variables correctly classified 87 to 100% of cases. For comparison, 10 different LDA models were trained and tested using the same data sets with either the original data or with logarithmically transformed data. Again, half of the models used all 20 variables while the others used only the five significant variables. LDA models provided correct classifications in 62.5% to 87.5% of cases. The classifications provided by all of the different models were compared with kappa statistics, yielding kappa values ranging from 0.25 to 1.0. We conclude that ANN models based on DNA methylation profiles can objectively classify SCLC and NSCLC cells lines with substantial to perfect concordance, while LDA models based on DNA methylation profiles provide poor to substantial concordance. Our work supports the promise of ANN analysis of DNA methylation data as a powerful approach for the development of automated methods for lung cancer classification.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter.

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

5' Flanking Region↗
PubMed1992

Further studies on N-methyl-1(3,4-methylenedioxyphenyl)-2-aminopropane as a discriminative stimulus: antagonism by 5-hydroxytryptamine3 antagonists.

Using a standard two-lever operant paradigm, male Sprague-Dawley rats were trained to discriminate 1.5 mg/kg N-methyl-1(3,4-methylenedioxyphenyl)-2- aminopropane (MDMA) from saline using a variable-interval 15-s schedule of reinforcement for food reward. Tests of stimulus antagonism were conducted to further define the mechanism of action of MDMA as a discriminative stimulus. Low doses of the 5-hydroxytryptamine1A (5-HT1A) antagonist NAN-190, the 5-HT2 antagonist pirenperone, and the dopamine antagonist haloperidol were able to somewhat attenuate the MDMA stimulus; however, none of these agents decreased MDMA-appropriate responding to less than 46%. The 5-HT3 antagonists zacopride and LY 278584 (ID50 = 0.02 micrograms/kg) antagonized the MDMA discriminative stimulus. Zacopride also attenuated the stimulus effects of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) in DOM-trained animals but not those of (+)amphetamine in (+)amphetamine-trained animals. Several possible mechanistic interpretations are provided but it is concluded that MDMA produces its stimulus effects via a complex mechanism involving both dopaminergic and serotonergic components.

3,4-Methylenedioxyamphetamine↗