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PubMed2025

Comprehensive assessment of homologous recombination deficiency via simultaneous methylation and mutation analysis in epithelial ovarian cancer: implications for PARP inhibitors efficacy.

BACKGROUND: The advent of poly (ADP-ribose) polymerase inhibitors (PARPi) over the past decade has significantly altered the management of epithelial ovarian cancer (EOC). We proposed that the etiology of homologous recombination deficiency (HRD) might underlie the variable responses to PARPi observed across patient populations. METHODS: As part of the phase 2 study of the Chinese HRD Harmonization Project, we developed a genomic methylation sequencing (GM-seq) pipeline facilitated by the TET enzyme for the simultaneous identification of methylated modifications and genetic variations in EOC tumor samples, and compared with established DNA sequencing-based HRD assays. RESULTS: Somatic mutation and HRD scores were confounded by low tumor purity in our cohort of 98 locally advanced/advanced EOC patients. In samples with tumor purity ≥ 30% (n = 45), the GM-seq pipeline showed high consistency with DNA sequencing-based HRD assay, identifying genetic variations in homologous recombination repair (HRR) genes and HRD score with 92.6% (25/27) and 97.1% (33/34) consistency respectively, in addition to conducting methylation profiling. Moreover, different underlying mechanisms of HRD were associated with varying degrees of PARPi efficacy, with BRCA1/2 LOH group having the best efficacy (median PFS, undefined), followed by BRCA1 methylation group (median PFS, 23.4 months), and those with unknown etiology of HRD having the worst efficacy (median PFS, 8.8 months, p < 0.001). CONCLUSION: Our findings underscore the importance of considering HRD etiology when evaluating PARPi efficacy in EOC patients. The GM-seq pipeline, represents a significant advancement in HRD detection, enabling more accurate predictions of PARPi response.

Epithelial ovarian cancer (EOC)↗

PubMed1996

Cytological evidence for 5-azacytidine-induced demethylation of the heterochromatic regions of human chromosomes.

This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.

5-Methylcytosine↗

PubMed1997

Effect of exogenous and endogenous nitric oxide on the airway and tissue components of lung resistance in the newborn piglet.

Despite widespread reports of the vasodilatory actions of nitric oxide (NO), little is known of the relaxant effect of NO on newborn airways or lung parenchymal structures. We studied the effects of inhaled NO at 20, 40, and 80 ppm on lung (Rl), tissue (Rti), and airway (R(aw)) resistance in 13 2-5-d-old anesthetized, ventilated, open-chested piglets. Rl was measured from transpulmonary pressure and air flow. Rti was measured by alveolar capsules, and R(aw) was calculated as the difference between Rl and Rti. Any given concentration of inhaled NO (20, 40, or 80 ppm) significantly decreased Rl (p < 0.001), Rti (p < 0.001), and R(aw) (p < 0.05). In addition, blockade of endogenous NO with 30 mg/kg N omega-nitro-L-arginine methyl ester (L-NAME) given i.v. in 12 piglets significantly increased Rti and Rl with variable changes in R(aw), and caused a decrease in dynamic compliance. Readministration of NO to eight piglets induced a significant decreased in Rl and Rti at 20 and 80 ppm, whereas R(aw) significantly decreased only at 80 ppm. Pulmonary arterial pressure decreased after exposure to inhaled NO and increased after L-NAME administration. Systemic arterial pressure was unaffected by inhaled NO but increased after L-NAME administration. Our results indicate that Rl, R(aw), and Rti are reduced by exogenous NO, suggesting NO-mediated airway smooth muscle relaxation throughout the newborn lung. In contrast, blockade of endogenous NO significantly increases only Rti, suggesting a physiologic role for endogenous NO in regulation of peripheral contractile elements. We speculate that NO-mediated modulation of resistance in pulmonary parenchyma may serve to regulate the balance of ventilation and perfusion and resultant gas exchange in the lungs during early postnatal development.

Airway Resistance↗

PubMed1992

The clinical efficacy of single morning doses of levodopa methyl ester: dispersible Madopar and Sinemet plus in Parkinson disease.

For many patients with Parkinson disease and levodopa-related motor fluctuations, the latency to onset of action of a single dose of a levodopa preparation may be both long and variable. In an effort to find a more rapidly acting and reliable preparation of levodopa, we therefore studied the efficacy of single doses of an oral solution of 250 mg of levodopa methyl ester (ME) with benserazide, 50 mg and of a molar equivalent dose of dispersible Madopar (DM) (50/200) in 13 patients in the fasting state after overnight drug withdrawal. The response of seven of these patients was compared to that after two Sinemet 25/100. The latency to "on" was equally fast with ME and DM, and significantly faster than after standard Sinemet. The duration of "on" was similar with all three. Because of this more rapid relief of "off" periods, both ME and DM offer a potential clinical advantage over standard preparations of levodopa.

Benserazide↗

PubMed2002

Rat motoneuron cell death in development correlates with loss of N-methyl-D-aspartate receptors.

New techniques were applied for maintaining viable motoneurons in rat cervical spinal cord slices to study electrical and morphological properties from postnatal day (PD) 2-49. Lucifer Yellow injections showed nine to 12, or more, viable motoneurons/slice at PD2, reduced to two to three in lamina IX by PD9. At PD2 and from PD14 onward healthy motoneurons were electrically similar to those of adults. Motoneurons exhibited variable electrical properties and morphology around PD5. They were sensitive to kainate and AMPA at all ages. The sensitivity to N-methyl-D-aspartate (NMDA) was significant at PD2, less at PD9 and virtually absent at PD14. Our observations suggest that NMDA receptors play a role in regulation of motoneuron survival in the early postnatal period, but are lost from adult motoneurons.

Age Factors↗

PubMed2005

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.

Aeromonas↗

PubMed2001

p16INK4A-alterations in primary angiosarcoma of the liver.

BACKGROUND/AIMS: Alterations in the p16 (CDKN2/MTS-1/INK4A) gene have been implicated in the tumorigenesis of different human cancers. Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is the predominant mechanism of p16INK4a gene inactivation in malignant epithelial tumors. This study was performed to determine whether alterations of p16 are involved in the development of angiosarcoma of the liver. METHODS: The status of p16 was evaluated in 17 angiosarcomas of the liver by methylation-specific PCR (MSP), microsatellite analysis, DNA sequencing and immunohistochemical staining. The results obtained were correlated with histopathological variables and with patient survival. RESULTS: Hypermethylation of the 5' CpG island of the p16 gene was found in 12 out of 17 (71%) angiosarcomas examined. Homozygous deletion at the p16 region was present in one case (6%), and loss of heterozygosity was present in two cases (12%). We failed to detect p16 gene missense mutations. The status of p16 correlated with neither histopathological factors nor with the prognosis of the patients with angiosarcomas. CONCLUSIONS: These data suggest that inactivation of the p16 gene is a frequent event in angiosarcomas of the liver. The most common somatic alteration is promotor methylation of the p16 gene. We failed to establish p16 as independent prognostic factors in these tumors.

Aged↗

PubMed1994

Cardiovascular effects of basic fibroblast growth factor in rats.

We determined the effects of human basic fibroblast growth factor (bFGF) on blood pressure (BP) and heart rate (HR) in pentobarbital-anesthetized rats and examined the possible involvement of nitric oxide (NO) and prostanoids in these effects. Intravenous (i.v.) injections of bFGF (1, 7.5, and 15 micrograms/kg) induced dose-dependent short-lasting decreases in BP followed by a striking increase in BP and HR variability. The BP and HR increases in variability were closely related (r = 0.95; n = 20; p < 0.001). Pretreatment with a single intravenous (i.v.) dose of N omega-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg) almost suppressed (-90%) the cardiovascular effects of bFGF, an effect that was restored by a single dose of L-arginine (100 mg/kg i.v.). Similar suppression was observed with use of indomethacin (10 mg/kg i.v.). Indomethacin given 50-60 min after bFGF abolished the increased variability of BP and HR. We conclude that the BP decrease and the increase in BP and HR variability induced by bFGF involve release of both NO and vasoactive prostanoids.

Animals↗

PubMed1997

Cloning and characterization of the gene encoding PspPI methyltransferase from the Antarctic psychrotroph Psychrobacter sp. strain TA137. Predicted interactions with DNA and organization of the variable region.

The gene (pspPIM) encoding the PspPI DNA methyltransferase (MTase) associated with the PspPI restriction-modification (R-M) system (5'-GGNCC-3') of Psychrobacter species TA137 has been cloned and expressed in E. coli, and its nucleotide (nt) sequence has been determined. The coding region was 1248 nt in length and capable of specifying a 46826-Da protein of 416 amino acids (aa). The predicted sequence of the MTase protein displays ten sequence motifs characteristic of all prokaryotic m5C-MTases and shows the highest similarity to other MTases that methylate the GGNCC sequence, namely M . Eco47II and M . Sau96I. All three MTases methylate the internal cytosine within their recognition sequence. Sequence similarities between M . PspPI and its isospecific M . Eco47II and M . Sau96I as well as with four other m5C-MTases that methylate the related GGWCC sequence, namely M . SinI, M . HgiCII, M . HgiBI, M . HgiEI have been also found within the variable region of these proteins. On the basis of structural information from M . HhaI and M . HaeIII, several M . PspPI residues that are expected to interact with DNA can be predicted. Furthermore, an organization of the variable region of m5C-MTases into two segments exhibiting a pattern of conserved residues and a considerable degree of structural homologies is described.

Amino Acid Sequence↗

PubMed1979

[Polymorphism of a tumor ce-l population and selection processes. IV. The effect of dibunol and nitrosourea on the variability of Ehrlich-I. Ch. Ph. ascitic strain tumor cells].

A study was made of the effect of dibunol and methyl-N-nitrosourea (MNU) on two tumor cell subpopulations of the Ehrlich-I. Ch. Ph. ascites strain, one of which is characterized with A + B + 2C and A + D + 2C--markers and the other one--with A1 + A2 + 2B + D + C markers. Dibunol that belongs to the class of inhibitors of free-radical processes was shown to bring about changes in cell subpopulations, the mode of changes depending on the dose and regime of treatment. The effect of MNU on the population resulted predominantly in the accumulation of cells with various chromosome aberrations. At early stages of tumor progression, aberrations were more pronounced in cells with marker chromosome "A" than in the cells with 44 chromosomes and markers A1 + A2 + 2B + D + C.

Animals↗

PubMed1995

Contribution of O6-methylguanine-DNA methyltransferase to resistance to 1,3-(2-chloroethyl)-1-nitrosourea in human brain tumor-derived cell lines.

To assess the possible role of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in resistance of brain neoplasms to the clinically important chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), we quantitated MGMT activity, BCNU survival, and the effect of ablating MGMT activity on the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines. BCNU resistance, measured as 10% survival dose (LD10), differed eightfold among the lines. Elimination of measurable MGMT activity with the substrate analogue inhibitor O6-benzylguanine (O6-BG) revealed a variable but limited contribution of MGMT to survival. In no case did O6-BG reduce LD10 by more than 3.4-fold. In contrast, O6-BG reduced the LD10 for N-methyl-N'-nitro-N-nitrosoguanidine up to 31-fold in the same cell lines (Bobola MS, Blank A, Berger MS, Silber JR, Mol Carcinog 13:70-80, 1995). Variability in BCNU survival, manifested as a sevenfold range of LD10, persists after measurable MGMT was eliminated, indicating that another mechanism or mechanisms is operating to limit cytotoxicity. Cells alkylated while suspended in growth medium are more resistant to BCNU and display less dependence on MGMT than cells treated while proliferating on a plastic substratum. When alkylated in suspension, most of the lines are either unresponsive to O6-BG or contain a subpopulation that did not respond to O6-BG. Our results demonstrate that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in our lines.

Brain Neoplasms↗

PubMed1998

Neuroprotective effects of NPS 846, a novel N-methyl-D-aspartate receptor antagonist, after closed head trauma in rats.

OBJECT: The authors sought to determine whether 3,3-bis (3-fluorophenyl) propylamine (NPS 846), a novel noncompetitive N-methyl-D-aspartate receptor antagonist, alters outcome after closed head trauma in rats. METHODS: The experimental variables were: presence or absence of closed head trauma, treatment with NPS 846 or no treatment, and time at which the rats were killed (24 or 48 hours). The NPS 846 (1 mg/kg) was administered intraperitoneally at 1 and 3 hours after closed head trauma or sham operation. Outcome measures were the neurological severity score (NSS), ischemic tissue volume, hemorrhagic necrosis volume, and specific gravity, water content, and concentrations of calcium, sodium, potassium, and magnesium in brain tissue. The following closed head trauma-induced changes in the injured hemisphere (expressed as the mean +/- the standard deviation) were reversed by NPS 846: decreased specific gravity of 1.035 +/- 0.006 at 24 hours was increased to 1.042 +/- 0.004; the decreased potassium level of 0.583 +/- 0.231 mg/L at 48 hours and at 24 hours was increased to 2.442 +/- 0.860 mg/L; the increased water content of 84.7 +/- 2.6% at 24 hours was decreased to 79.8 +/- 2%; the increased calcium level of 0.592 +/- 0.210 mg/L at 24 hours was decreased to 0.048 +/- 0.029 mg/L; and the increased sodium level of 2.035 +/- 0.649 mg/L was decreased to 0.631 +/- 0.102 mg/L. Administration of NPS 846 also lowered the NSS (improved neurological status) at 48 hours (7 +/- 3) and caused no significant changes in ischemic tissue or hemorrhagic necrosis volumes in the injured hemisphere at 24 or 48 hours. CONCLUSIONS: In this model of closed head trauma, NPS 846 improved neurological outcome, delayed the onset of brain edema, and improved brain tissue ion homeostasis.

Animals↗

PubMed1990

Excitatory amino acid pathways in brain-stimulation reward.

A range of agonists and antagonists active at different glutamate/aspartate (Glu/Asp) receptor subtypes were injected into rat ventral tegmental (VTA) sites downstream from self-stimulation electrodes in the medial forebrain bundle. Control injections were made into the contralateral tegmentum. Variable-interval (VI 10 s) self-stimulation was not significantly affected by a specific antagonist of N-methyl-D-aspartate (NMDA)-type receptors (D,L-2-amino-5-phosphonovaleric acid (2-AP5), 10 and 50 nmol). Broad-spectrum excitatory amino acid (EAA) antagonists viz cis-2,3-piperidine dicarboxylate (cPDA) (10 and 50 nmol), gamma-D-glutamylaminomethyl sulphonic acid (GAMS) (10 nmol) and p-chlorobenzoyl-2,3-piperazine dicarboxylic acid (pCB PzDA) (2.0 and 10 nmol), active at kainate, quisqualate, as well as NMDA receptors, all produced significant depression of responding when injected into the ipsilateral, but not the contralateral, tegmentum. Compounds inhibiting Glu/Asp reuptake had variable effects: strong depression with dihydrokainic acid (7.5 nmol), or no significant effect (L-threo-3-hydroxyaspartic acid, 2.0 and 10 nmol). The receptor agonist, NMDA (10 nmol), depressed responding regardless of injection side; kainic and responding regardless of injection side; kainic and quisqualic acid elicited myoclonic and other non-specific responses in preliminary tests, and were not examined further; enhanced responding was not seen. The side-specific blockade of responding by non-NMDA antagonists indicates the existence of non-NMDA EAA terminals in the VTA, signalling the receipt of hypothalamic brain-stimulation reward. Caudally directed EAA projections terminating on A10 dopamine cell bodies may account for depression of self-stimulation by EAA antagonists.

Amino Acids↗

PubMed2004

Sterically hindered triacylglycerol analogues as potent inhibitors of human digestive lipases.

A novel class of inhibitors of human digestive lipases have been developed. Various sterically hindered triacylglycerols based on 2-methyl- and 2-butylglycerol, and/or 2-methyl fatty acids were synthesized. The triacylglycerol analogues were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface-pressure/molecular-area compression isotherms. The inhibition of human pancreatic and gastric lipases by the sterically hindered triacylglycerol analogues was studied by using the monolayer technique with mixed films of 1,2-dicaprin, which contained variable proportions of each inhibitor. Triolein analogues that contain a butyl group at the 2-position of the glycerol backbone or methyl groups both at the 2-position of glycerol, and the alpha-position of each oleic acid residue were potent inhibitors; this caused a 50% decrease in HPL activity at 0.003 molar fraction.

Enzyme Inhibitors↗

PubMed1994

Persistent increase of hippocampal presynaptic axon excitability after repetitive electrical stimulation: dependence on N-methyl-D-aspartate receptor activity, nitric-oxide synthase, and temperature.

The electrical excitability of Schaffer collateral axons and/or terminals was studied in hippocampal slices by monitoring single, CA3 pyramidal neurons activated antidromically from CA1 stratum radiatum. At 22 degrees C, weak, repetitive stimulation with as few as 10 impulses at 2 Hz led to a robust lowering of the antidromic activation threshold that lasted > 30 min. The effect was completely absent at 32 degrees C and was blocked by both the N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate and the inhibitor of nitric-oxide synthase, L-nitro-arginine methyl ester. Such threshold lowering would alter the variance of synaptic responses from axons stimulated in the variable excitation region of their input-output functions. These results thus raise important doubts about the interpretation of experiments in which the so-called minimal-stimulation method has been used at reduced temperature to infer changes in quantal transmission during hippocampal long-term potentiation. In the present experiments, no changes were observed in the estimate of excitatory postsynaptic potential quantal content in long-term potentiation experiments at either temperature, which could not be accounted for by an artificial, temperature-dependent change in the responsiveness of presynaptic axons.

Amino Acid Oxidoreductases↗

PubMed2005

Synthesis and conformational analysis of 6-C-methyl-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosyl mono- and disaccharides.

Several 6-C-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosides (beta-D-GlcNAc monosaccharides 1a-3a and 1,4-linked disaccharides 1b-3b) were studied by solution NMR spectroscopy. Conformational analysis of the (6S)- and (6R)-C-methyl-substituted beta-d-GlcNAc monosaccharides indicates that the stereodefined methyl groups impose predictable conformational biases on the exocyclic C-5-C-6 bond, as determined by (1)H-(1)H and (13)C-(1)H coupling constants. Variable-temperature NMR experiments in methanol-d(4) were performed to determine DeltaDeltaH and DeltaDeltaS values derived from the two lowest energy conformers. These indicate that while the influence of 6-C-methyl substitution on conformational enthalpy is in accord with the classic principles of steric interactions, conformational preference in solution can also be strongly affected by other factors such as solvent-solute interactions and solvent reorganization.

Algorithms↗

PubMed2002

Angelman syndrome methylation screening of 15q11-q13 in institutionalized individuals with severe mental retardation.

Among several genetic diseases that comprise mental retardation, Angelman syndrome (AS) has been extensively recognized and investigated. In the general population, the syndrome occurs in about 1 in 20,000 live births and its prevalence in severely mentally retarded individuals is 1.4%. These figures, however, may be an underestimate, because of the variable phenotype of AS. The main objective of this work was to investigate AS patients among a group of mentally retarded subjects, using the methylation pattern of the SNRPN gene, as determined by Southern blotting molecular analysis. The molecular investigation of 75 institutionalized individuals with severe to profound mental retardation resulted in the detection of 1 case with an abnormal methylation pattern of the SNRPN gene, corresponding to AS. The patient's phenotype was classified as atypical, without outbursts of inappropriate laughter or a happy disposition; the patient would not have been diagnosed in the usual screens for AS, which only select patients who demonstrate the typical clinical findings characteristic of the disease.

Angelman Syndrome↗

PubMed2003

Biogeochemistry of mercury in the Amazonian environment.

In this paper, the processes that affect mercury (Hg) cycling in the Amazonian environment were reviewed, criticized and new directions of research are proposed. The discussion of the origin of the mercury contamination, whether natural or anthropogenic is marked by a lack of fundamented arguments from both sides. Undoubtedly mercury inputs from gold mining have locally increased environmental concentrations, but in the whole Amazon, these loads would be insignificant, considering the high concentrations observed by some authors in remote soils. A reasonable process that should explain these elevated concentrations in soil is that B horizons function as a mercury "sponge" that have been accumulating mercury over a geological time scale, releasing it back to cycling during erosion and forest fires. The environmental degradation of the Amazonian forest due to human activities is probably enhancing the release of that mercury to the cycle. Mercury transformations in reduced, anoxic environments--sediments and waters--are also a key problem for the understanding of the environmental methylation. The studies that have been carried out in the Amazonian environment are too restricted and results permit only circumstantial conclusions. Large efforts must be directed to monitoring programs considering time and space variability.

Alkylation↗