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Results for “variable methylation”

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PubMed1987

Intra-subject variability of platelet alpha 2-adrenoceptor binding sites in healthy volunteers.

The specific alpha 2-adrenoceptor binding (Bmax and KD) of (3H-methyl)yohimbine to intact platelets of healthy young volunteers was not significantly different in 7 males and 7 females. Its extent was not dependent on the menstrual cycle. The intraindividual variability was relatively high with means between 6% and 38% for Bmax and between 10% and 39% for KD, respectively. An intra-assay variation of 5.4 to 12.4% for Bmax and of 5.1 to 19.6% for KD contributed to this variability. Therefore, drug effects on platelet alpha 2-adrenoceptor binding in intraindividual cross-over studies are evidential only if the drug-induced changes exceed the intra-subject variability. This should be examined by at least 3 estimates of pre-drug values.

Adult↗

PubMed1999

Induction of ribosome methylation in MLS-resistant Streptococcus pneumoniae by macrolides and ketolides.

One major mechanism for resistance to macrolide antibiotics in Streptococcus pneumoniae is MLS (macrolide, lincosamide, and streptogramin B) resistance, manifested when the 23S rRNA is methylated by the product of an erm gene. This modification results in the decreased binding of all known macrolide, lincosamide, and streptogramin B antibiotics to the ribosome. More than 30 ermAM-containing clinical isolates of S. pneumoniae were examined in our lab and showed high-level resistance (MIC > or =128 microg/ml) to erythromycin, azithromycin, tylosin, clindamycin, and ketolide (macrolides that lack the cladinose sugar) TE-802. We found that the new generation of ketolides A965 and A088 displayed variable activity against the same group of resistant S. pneumoniae strains. To understand the basis of variability of the minimal inhibitory concentration (MIC) values of A965 and A088, we examined the effects of a series of macrolides and ketolides on the level of 23S rRNA methylation in five ermAM-containing resistant S. pneumoniae isolates. We show here that the basal levels of ribosomal methylation vary from strain to strain. The level of rRNA methylation can be strongly induced by erythromycin, azithromycin, and TE-802, resulting in high-level of resistance to these compounds. Ketolide A965 and A088, however, are weak inducers at sub-MIC drug concentrations, therefore showing variable activities in strains with differential methylation levels.

Anti-Bacterial Agents↗

PubMed1992

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact.

Base Sequence↗

PubMed2003

Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients.

The newly identified 3p21.3 tumour suppressor gene RASSF1A is methylated in the majority of primary lung tumours, lung tumour cell lines and in a variable percentage of breast tumours. To determine the extent of RASSF1A promoter hypermethylation in early lung tumorigenesis, we analysed sputum samples from lung cancer patients and from current and former smokers using a sensitive methylation-specific PCR (MSP) technique. We also analysed RASSF1A promoter region hypermethylation in trios of normal breast/invasive ductal breast carcinoma/ductal carcinoma in situ (DCIS) from breast cancer patients and DCIS without invasive cancer. We found that 50% of small cell lung cancer (SCLC) and 21% of non-small cell lung cancer (NSCLC) patients had RASSF1A methylation, while one of two former smokers and four of 13 current smokers demonstrated RASSF1A methylation in sputum. Furthermore, two of the four current smokers and one former smoker showing RASSF1A methylation in their sputum developed cancer within 12-14 months of bronchoscopy. In our breast cancer trios, RASSF1A promoter hypermethylation was detected in 65% of invasive cancers, in 42% of corresponding DCIS but in none of the normal breast samples. In addition, we found that three out of 10 DCIS without invasive breast cancer also underwent RASSF1A promoter hypermethylation. Our findings suggest that RASSF1A promoter region hypermethylation may be a useful molecular marker for early detection of lung cancer. Furthermore, since RASSF1A promoter hypermethylation was detected in ductal carcinoma in situ, inactivation of RASSF1A may be an early event in breast tumorigenesis.

Base Sequence↗

PubMed1983

Variable 5-methylcytosine levels in human tumor cell lines and fresh pediatric tumor explants.

DNA methylation was measured in human tumor cell lines and freshly explanted pediatric tumors by [3H]uridine labeling followed by DNA hydrolysis and high-performance liquid chromatography. The percentage of cytosine residues modified to 5-methylcytosine in 4 cell lines derived from pediatric tumors was increased or similar to the levels of 3.45% found in two strains of diploid human fibroblasts. However, a fibrosarcoma line derived from an adult (HT1080) contained decreased levels of 5-methylcytosine. Four low-passage retinoblastomas had uniform 5-methylcytosine levels of approximately 3.9%, whereas the levels of the modified base in two freshly explanted rhabdomyosarcomas, one Wilms' tumor, and one medulloblastoma were 3.39, 3.56, and 3.45%, respectively. Two neuroblastomas had substantially lower levels (2.63 to 2.83%) of 5-methylcytosine in their DNAs compared to the retinoblastomas, which are also derived from neural tissue. The results show that while tumors of the same histological type had similar 5-methylcytosine values, discrepancies existed between values obtained for fresh tumors and their corresponding cell lines. Tumor cell lines may therefore be inadequate models for studies on the relationship between DNA methylation and cancer. The results also indicate that generalized statements on DNA methylation levels in pediatric tumors cannot be made, although substantial variations may occur between different tumors types.

5-Methylcytosine↗

PubMed1982

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture.

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2'-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10(-6) M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10(-5) M of each analog. In the presence of 10(-6) M 2'-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxy-cytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [3H]formycin and [3H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2'-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [3H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.

Adenosine↗

PubMed2000

Hypermethylation of the p16 (Ink4a) promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines.

Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16 (Ink4a) tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (>/=50% methylation at two or more of the CpG sites examined) of the p16 (Ink4a) promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16 (Ink4a) gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16 (Ink4a) expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Re-expression of p16 (Ink4a) and partial demethylation of the p16 (Ink4a) promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.

Adenocarcinoma↗

PubMed2002

Structural effects of cytosine methylation on DNA sugar pucker studied by FTIR.

This FTIR investigation concerns structural consequences of 5-methylation of cytosine in a DNA decamer in solution. Methylation of DNA is an important functional signal in transcription, but its effect on DNA structure is variable and not fully understood. Here, single and multiple 5-methylcytosine substitutions are introduced into the self-complementary sequence d(CCGGCGCCGG)(2). No major structural effect of methylation on the DNA duplex in solution is seen in the IR spectra: The overall B-form character of the backbone and S-type of sugar puckering are maintained in all the studied sequences, in agreement with previous literature. However, certain significant effects are detected in the IR regions sensitive to sugar pucker and glycosidic torsional angle. A single or multiple 5-methylcytosine substitution in d(CCGGCGCCGG)(2) leads to a doublet splitting of the S-type 840-820 cm(-1) sugar conformational band. The results suggest the coexistence of two different major sugar puckers within the S-conformational family, with an increased relative contribution of the C2'-endo type of sugar in the methylated sequences. In addition, a partial or full downshift of the guanosine/anti marker band at 1,375 cm(-1) in the methylated sequences reflects a change in the value of the dihedral angle chi of guanosine upon methylation. The IR spectra are interpreted in terms of localized transitions between the BI and BII subconformational states of the B-DNA backbone caused by the methylation. An increased amount of the BII subconformer in the methylated sequences should give rise to a structurally more rigid conformation, in agreement with earlier observations on DNA backbone dynamics and bending flexibility in methylated DNA.

Base Sequence↗

PubMed2006

Decreased expression of insulin-like growth factor binding protein 7 in human colorectal carcinoma is related to DNA methylation.

PURPOSE: Insulin-like growth factor binding protein 7 (IGFBP-7) is considered a tumor suppressor in various cancers, but its role in colorectal cancer (CRC) is still uncertain. The aims of this study were to analyze the IGFBP-7 expression, and explore the mechanism responsible for the inactivation of IGFBP-7 in CRC. METHODS: mRNA expression was studied by RT-PCR and Northern blot analysis of cultured cells. Methylation status was analyzed by treatment with 5-aza-2'-deoxycytidine followed by sequencing of PCR products of sodium bisulfite-treated genomic DNA. IGFBP-7 protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays. RESULTS: mRNA expression was lost in six out of eight CRC cell lines as compared to normal colon cells. DNA methylation was found in the region of exon 1 and intron 1 of IGFBP-7. In tumor tissue, 107 out of 279 samples showed a negative expression of IGFBP-7 by IHC, which was significantly associated with poor prognosis. The analysis of 37 paired cancerous and normal mucosa samples confirmed the downregulation in the tumors, but revealed variable basal expression levels of IGFBP-7 in normal mucosal samples. CONCLUSIONS: DNA methylation is a mechanism responsible for IGFBP-7 gene silencing providing a target for therapeutic intervention of this tumor suppressor gene.

Cell Line, Tumor↗

PubMed2004

Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.

5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine (AC-3933) is a novel cognitive enhancer with central benzodiazepine receptor partial inverse agonistic activity. AC-3933 is predominantly metabolized to hydroxylated metabolite [SX-5745; 3-(5-hydroxymethyl-1,2,4-oxadiazol-3-yl)-5-(3-methoxyphenyl)-2-oxo-1,2-dihydro-1,6-naphthyridine] in dog. Initially, we found that there is considerable interindividual variability in AC-3933 hydroxylation in dogs and that dogs could be phenotyped as extensive metabolizer (EM) and poor metabolizer (PM). Then, to clarify the cause of AC-3933 polymorphic hydroxylation in dogs, in vitro studies were carried out using liver microsomes from EM and PM dogs. Our results show that AC-3933 hydroxylation clearance in PM dogs was much lower than that in EM dogs (0.2 versus 10.8-20.5 microl/min/mg, respectively). In addition, AC-3933 hydroxylation was significantly inhibited by alpha-naphthoflavone, a CYP1A inhibitor, and by anti-CYP1A2 antibodies, indicating that CYP1A2 was responsible for the polymorphic hydroxylation of AC-3933 in dogs. Furthermore, immunoblotting results have shown that although CYP1A2 protein was not detected in PM dogs (<0.86 pmol/mg), CYP1A2 content in EM dogs was prominent (6.1-13.0 pmol/mg). These results indicate that AC-3933 polymorphic hydroxylation arises from the polymorphic expression of CYP1A2 in dogs, which might involve genetic polymorphism of the CYP1A2 gene.

Administration, Oral↗

PubMed2005

Comprehensive analysis of CDKN2A status in microdissected urothelial cell carcinoma reveals potential haploinsufficiency, a high frequency of homozygous co-deletion and associations with clinical phenotype.

PURPOSE: There are significant differences in reported frequencies, modes of inactivation, and clinical significance of CDKN2A in urothelial cell carcinoma (UCC). We aimed to address these issues by investigating all possible modes of inactivation and clinicopathologic variables in a single tumor panel. EXPERIMENTAL DESIGN: Fifty microdissected UCCs were examined. CDKN2A gene dosage (quantitative real-time PCR), allelic status (microsatellite analysis), hypermethylation (methylation-specific PCR), mutation status (denaturing high-performance liquid chromatography and sequencing), protein expression (immunohistochemistry), and clinicopathologic variables (stage, grade, and disease recurrence during follow-up) were assessed. RESULTS: Exon 2 was underrepresented in 20 of 46 (43%) and exon 1beta in 21 of 46 (46%) of cases. Underrepresentation of exon 2 was accompanied by loss of heterozygosity (LOH) of 9p in 6 of 18 (30%) and of exon 1beta in 11 of 19 assessable cases (58%). Overall, LOH of 9p was identified in 15/41 (37%). Homozygous deletion of exons 2 and 1beta was detected in 16 of 46 (35%) and 10 of 46 tumors (22%), respectively. Co-deletion was most common, but exon 2-specific homozygous deletion was also detected. In tumors without homozygous deletion, p16 promoter hypermethylation was detected in 1 of 18 (6%). Hypermethylation of the p14ARF promoter or mutations in CDKN2A were not observed. Homozygous deletion of exon 2 or LOH on 9p were associated with invasion. Homozygous deletion of exon 2 or exon 1beta was associated with recurrent disease. CONCLUSIONS: These results confirm CDKN2A as a clinically relevant target for inactivation in UCC and show that the true frequency of alteration is only revealed by comprehensive analysis. Our results suggest that CDKN2A may be haploinsufficient in human cancer.

Chromosomes, Human, Pair 9↗

PubMed2005

Prognostic implication of aberrant promoter hypermethylation of CpG islands in adenocarcinoma of the lung.

OBJECTIVES: DNA hypermethylation in promoter regions has been studied for various types of cancer. However, there is no clear evidence that shows whether methylation status can predict long-term survival in patients with lung cancer. METHODS: We collected tissues from 72 patients with lung adenocarcinomas. The cancer and normal lung tissues were tested for DNA hypermethylation by using methylation-specific polymerase chain reaction. The genes investigated were p16INK4alpha(p16), retinoic acid receptor beta-promoter (RARbetaP2), death-associated protein kinase (DAPK), O6-methylguanine-DNA-methyltransferase (MGMT), and glutathione-S-transferase P1 (GSTP1). The status of the DNA methylation was analyzed, and we focused on long-term outcomes, as well as other clinical variables. RESULTS: DNA hypermethylation was observed in 83% for p16, 63% for RARbetaP2, 32% for DAPK, 17% for MGMT, and 46% for GSTP1 from the cancer tissue. From normal lung tissue, the results of methylation were positive in 75% for p16, 24% for RARbetaP2, 10% for DAPK, 6% for MGMT, and 33% for GSTP1. During the mean follow-up period of 18 +/- 11 months (1-40 months), 25 (35%) patients experienced recurrence, and 13 died. In multivariable analysis, old age (>60 years, P = .007), male sex (P = .004), unmethylation of DAPK from cancer tissue (P = .045), and hypermethylation of RARbetaP2 from normal tissue (P = .000) were risk factors for poor survival. Pathologic stage (P = .023), unmethylation of DAPK from normal tissue (P = .043), and hypermethylation of RARbetaP2 from normal tissue (P = .030) were risk factors for disease-free survival. CONCLUSIONS: DNA methylation status of CpG islands seems to be a useful predictor of long-term outcome for adenocarcinoma of the lung. However, because the predictive power is still low, further studies, including those with multiple genes, are necessary to increase its usefulness in the clinical setting.

Adenocarcinoma↗

PubMed1983

Acrylate and methacrylate esters: relationship of hemolytic activity and in vivo toxicity.

Quantitative hemolysis assays of acrylate and methacrylate esters provided estimates of the intrinsic hemolytic activity (Hi, the slope of the concentration-response curve) and the concentrations effecting 5% (H5) and 50% (H50) hemolysis. The dependence of hemolytic activity and LD50 (mice) on physical properties (lipophilicity, molar refraction, and molecular volume) of the esters was determined by multiple regression analysis. The observed correlations were: Hi, R2 = 0.94; H5, R2 = 0.95; H50, R2 = 0.94; and LD50, R2 (all compounds) = 0.80, R2 (all compounds less the methyl esters) = 0.94. The difference of the methyl esters was associated with the smaller steric volume of the methyl ester substituent and the presence (methacrylates) or absence (acrylates) of the branched methyl group. Associative steric contributions of the branched methyl group and the ester substituents were probably responsible for greater variability in the methyacrylate series. The results were consistent with the conclusion that the mechanism of the action of the esters is membrane mediated and relatively nonspecific and that in vivo biotransformation was not a significant factor. Also, long-term toxic liability of the esters may be more closely related to intrinsic toxicity than acute toxicity.

Acrylates↗

PubMed1989

Three-dimensional structure of a fluorescein-Fab complex crystallized in 2-methyl-2,4-pentanediol.

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

Antibodies, Monoclonal↗

PubMed1988

Enzyme activity and behavior in hyperactive children grown up.

Thirty-one men, originally seen as children for problems with overactivity, distractibility, and impulsiveness, were reevaluated at ages 21-23 years. Subjects were interviewed and tested and provided a blood sample for analyses of dopamine-beta-hydroxylase (DBH), monoamine oxidase (MAO), and catechol-O-methyl-transferase (COMT). A series of Pearson correlations between the biological variables and psychological measures revealed that MAO levels were negatively associated with measures of drug involvement, cigarette use, fire-setting, and sensation seeking; DBH levels were positively associated with measures of sensation-seeking; and COMT levels were negatively associated with a measure of hostility and positively correlated with a measure of impulsiveness.

Adult↗

PubMed2001

Study of the polymerization of anthracene oil with AlCl3 by chromatography and related techniques.

The structure and composition of products from the reaction of anthracene oil with anhydrous AlCl3 have been examined. Size-exclusion chromatography has been carried out using a column with polystyrene-polydivinylbenzene as stationary phase, 1-methyl-2-pyrrolidinone at 80 degrees C as eluent and variable-wavelength UV-absorption detection. This system provides a chromatogram of the sample with several peaks. Molecular masses corresponding to these peaks were estimated using a calibration curve obtained with polycyclic aromatic hydrocarbon standards, ranging from 92 to 532 u. The most abundant compounds of the substrate were dimers and trimers of the main anthracene oil components. These results are corroborated on a qualitative level by synchronous UV-fluorescence spectra.

Aluminum Chloride↗

PubMed2002

Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution.

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 microg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 microg/ml-0.29 mg/ml). At concentrations below 0.9 microg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m7Guo) with a catalytic rate constant 1.3x10(-3) x s(-1) as compared with the rate of 38 s(-1) for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m7Guo, 70 s(-1), is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 microM-30 mM phosphate is consistent with only one dissociation constant for phosphate, K(d)=220+/-120 microM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 microM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.

Animals↗

PubMed2026

Toward the clinical application of long-read sequencing in repeat-expansion disorders.

Repeat-expansion disorders (REDs) are a mechanistically and clinically well-defined subgroup of rare diseases caused by the expansion of short tandem repeats (STRs). These expansions can exceed several kilobases and show complex features, such as noncanonical secondary structures, somatic instability, repeat interruptions and allele-specific methylation. These characteristics are highly relevant for understanding disease mechanisms, clinical variability, prognosis and potentially therapeutic decision-making, but cannot be fully resolved using traditional diagnostic methods or short-read sequencing technologies. By contrast, long-read sequencing (LRS) enables accurate investigation of STR complexity in a single assay, facilitates the discovery of new pathogenic repeat expansions and drives advances in diagnostics, clinical and basic research, which may allow for better patient stratification in future clinical trials. This Perspective discusses recent LRS-driven discoveries, methodological and bioinformatic advances, and emerging diagnostic applications to illustrate the potential of LRS in reshaping both research and clinical practice.

Humans↗