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Biomedical subjects

M Ehrlich

Publications and source records attributed to M Ehrlich.

At least 109 records · Page 6Linked to original sources
PubMed1989

Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from mammalian cells binds specifically to six pBR322 and M13mp8 DNA sequences but only when they are methylated at their CpG dinucleotide pairs. We cloned three high-affinity MDBP recognition sites from the human genome on the basis of their binding to MDBP. These showed much homology to the previously characterized prokaryotic sites. However, the human sites exhibited methylation-independent binding apparently because of the replacement of m5C residues with T residues. We also identified three other MDBP sites in the herpes simplex virus type 1 genome, two of which require in vitro CpG methylation for binding and are in the upstream regions of viral genes. A comparison of MDBP sites leads to the following partially symmetrical consensus sequence for MDBP recognition sites: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3'; m5Y (m5C or T), R (A or G), Y (C or T). This consensus sequence displays an unusually high degree of degeneracy. Also, interesting deviations from this consensus sequence, including a one base-pair deletion in the middle, are sometimes observed in high-affinity MDBP sites.

Base Sequence↗
PubMed1989

Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and dopamine DA1 agonist-sensitive Na+,K+-ATPase in renal tubule cells.

The cellular localization of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 that appears to mediate certain actions of dopamine in the mammalian brain by acting as an inhibitor of protein phosphatase 1, was studied in the kidney of several species. DARPP-32 mRNA and DARPP-32-like immunoreactivity were found in the cytoplasm of cells in the thick ascending limb of the loop of Henle. The specific dopamine DA1 agonist SKF 82526 caused a dose-dependent inhibition of Na+,K+-ATPase activity, which could be blocked by SCH 23390, a specific DA1 antagonist, and by PKI-(5-24) amide, a specific inhibitor of cAMP-dependent protein kinase. The results indicate that DA1 dopamine receptors and DARPP-32, an intracellular third messenger for dopamine, are part of the signal-transduction process for dopamine acting on renal tubule cells.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1989

Four cases of keratoconus and posterior polymorphous corneal dystrophy.

We examined four young men with keratoconus and posterior polymorphous corneal dystrophy. All four patients showed central corneal steepening and irregularity, and large areas of irregular polymorphous opacification at the level of Descemet's membrane (in at least one cornea of each patient), which are consistent with posterior polymorphous dystrophy.

Adult↗
PubMed1989

A plant DNA-binding protein that recognizes 5-methylcytosine residues.

A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.

5-Methylcytosine↗
PubMed1989

Pseudomonas corneal ulcer after use of extended-wear rigid gas-permeable contact lens.

We report a bacterial corneal ulcer that occurred in a patient who had worn extended-wear rigid gas-permeable contact lenses on a 1-week cycle for 9 months. Pseudomonas aeruginosa was cultured not only from the corneal ulcer but also from the contact lens solutions, suggesting that the patient had been noncompliant with care procedures. To our knowledge, this is the first report of a bacterial corneal ulcer associated with use of a rigid gas-permeable contact lens on an extended-wear basis.

Atropine↗
PubMed1989

Successful clonazepam treatment of neuroleptic-induced akathisia in older adolescents and young adults: a double-blind, placebo-controlled study.

In a double-blind, placebo-controlled trial of clonazepam in the treatment of neuroleptic-induced akathisia in older adolescents, clonazepam was significantly more effective at reducing akathisia scores at the end of the first treatment week than placebo (p less than 0.0001). Clonazepam may be a safe and effective treatment for neuroleptic-induced akathisia in this age group.

Adolescent↗
PubMed1988

Human methylated DNA-binding protein. Determinants of a pBR322 recognition site.

Methylated DNA-binding protein (MDBP) from human placenta has a high affinity for a site in pBR322 (pB site 1) when that site is methylated at its CpG dinucleotides. Dimethyl sulfate interference analysis and experiments with ligands prepared by oligonucleotide-directed mutagenesis indicate that 15 contiguous base pairs, 14 of which exhibit hyphenated dyad symmetry, influence MDBP binding to pB site 1. These 14 base pairs, 5'-RTMGYCAMGG(M/T)GAY-3' (M, 5-methylcytosine), suffice for recognition by MDBP as demonstrated with a double-stranded, MpG-containing oligonucleotide used as a free ligand or cloned into M13mp19 and subsequently methylated. Seven single-site mutations at different positions of this 14-base pair region largely eliminated binding, and several others increased binding up to 2-fold when compared to the nonmutant, triply methylated sequence. However, MDBP recognizes a site in hemimethylated M13mp19 replicative form DNA, which was homology to pB site 1 at only 10 of 14 base pairs, and all four of these different base pairs are equivalent to transversions. Based upon the above data, a mixed oligonucleotide probe was constructed that contains variants of pB site 1 which should be recognized by MDBP. This 14-base probe hybridizes under stringent conditions to a number of discrete fragments in restriction digests of human DNA. this suggests that there are multiple pB site 1-related sequences in human DNA that might, when methylated, bind MDBP in vivo.

Amino Acid Sequence↗
PubMed1988

Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

5-Methylcytosine↗
PubMed1988

Reduced genomic 5-methylcytosine content in human colonic neoplasia.

DNA methylation appears to play an important role in both physiological and experimentally modified gene expression, and alterations in DNA methylation have been described in animal tumor models and in transformed cells and tumor cell lines. However, there have been comparatively few reports on DNA methylation in primary human malignancies, and these reports are somewhat contradictory. While individual genes have shown hypomethylation in colon cancer and premalignant adenomas as well as in lung cancer, other genes have shown increased methylation, and absolute measures of 5-methylcytosine content have shown decreases in malignancies but not in premalignant adenomas. We have used a sensitive quantitative measurement of 5-methylcytosine content by high performance liquid chromatography revealing an unequivocal hypomethylation of tumor DNA. An average of 8 and 10% reduction in genomic 5-methylcytosine content was seen in apparently all colon adenomas and adenocarcinomas, respectively, and there was no significant difference between benign and malignant tumors. This is a substantial quantitative alteration and suggests a pervasive abnormality in the control of DNA methylation. Surprisingly, three patients with the highest 5-methylcytosine content in their normal colon appear to have a germline predisposition to cancer (Lynch syndrome).

5-Methylcytosine↗
PubMed1988

Difficulties encountered with some intermediate-atomic-number radiation-protection dosimeters irradiated on-phantom in low-energy photon beams.

During a measurement-assurance study done with a particular type of radiation-protection dosimeter, we obtained unexpectedly poor results when several of the dosimeters were irradiated simultaneously on-phantom in a low-energy bremsstrahlung beam. The dosimeters incorporated low- and intermediate-atomic-number (Z) thermoluminescence dosimeter elements with the intermediate-Z elements under Pb filters. The response of the low-Z elements was in good agreement with the predicted response regardless of dosimeter location on the phantom, while agreement for the intermediate-Z elements was poor for some locations not coinciding with the phantom center. We have since studied the behavior of this dosimeter type and of four other types in detail by irradiating them, one at a time, on- and off-phantom, with their geometric centers displaced up to 10 cm along the vertical and horizontal axes through the center of the beam cross section. Two low-energy bremsstrahlung beams and a 137Cs gamma-ray beam were used. Variation of response to low-energy photons with on-phantom location was observed to some degree in all intermediate-Z dosimeter elements covered with high-Z filters. The reason for the observed dependence of response upon on-phantom location seems to be insufficient side shielding against phantom and filter albedo, as proved in one case, in which added shielding removed this dependence for the element immediately adjacent to the added shield. Since the effective dose equivalent to the human body is lower for lateral incidence of low-energy photons than for the same photons incident from the front or back, dosimeters incorporating high-Z filters without side shielding (and thus expected to exhibit the phenomenon) do not lend themselves to the determination of the effective dose equivalent or any quantity proportional to it by currently employed methods. A detailed study of the angular dependence of dosimeter response performed on a suitable phantom should aid in eliminating such inadequate dosimeters from practical use.

Calibration↗
PubMed1987

Hypermethylation of human DNA sequences in embryonal carcinoma cells and somatic tissues but not in sperm.

Certain human DNA sequences are much less methylated at CpG sites in sperm than in various adult somatic tissues. The DNA of term placenta displays intermediate levels of methylation at these sequences (Sp-0.3 sequences). We report here that pluripotent embryonal carcinoma (EC) cells derived from testicular germ cell tumors are hypermethylated at the three previously cloned Sp-0.3 sequences and seven newly isolated sequences that exhibit sperm-specific hypomethylation. In contrast to their hypermethylation in EC cells, the Sp-0.3 sequences are hypomethylated in a line of yolk sac carcinoma cells, which like placenta, represent an extraembryonic lineage. These DNA sequences, therefore, appear to be subject to coordinate changes in their methylation during differentiation, probably early in embryogenesis, despite their diversity in copy number (1 to 10(4] and primary structure. Two of these Sp-0.3 sequences are highly homologous to DNA sequences in human chromosomal regions that might be recombination hotspots, namely, a cryptic satellite DNA sequence at a fragile site and the downstream region of the beta-globin gene cluster.

5-Methylcytosine↗
PubMed1987

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes.

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.

Bacteriophages↗
PubMed1987

N4-methylcytosine as a minor base in bacterial DNA.

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.

Bacteria↗
PubMed1986

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.

Base Sequence↗
PubMed1986

Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.

Base Sequence↗
PubMed1986

Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs.

Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.

Base Sequence↗
PubMed1986

Strain differences in distribution of phenylethanolamine N-methyltransferase activity from rat brain and adrenal gland.

Phenylethanolamine N-methyltransferase (PNMT) activity was measured in adrenal glands and medulla oblongata from 4 inbred rat strains, Fischer 344, Buffalo, Lewis and Sprague-Dawley rats. Adrenal enzyme activity was markedly different among the strains with the highest in Fischer, followed by Sprague-Dawley, Lewis and Buffalo rats in decreasing order. In medulla oblongata, the PNMT activity of Buffalo rat was the lowest being about one half of that of the other strains. Despite differences in the enzymes activity, immunotitration results indicate that there is no immunochemical difference between adrenal or medulla oblongata PNMT among the strains. Furthermore, the strain differences in the activity are not due to presence of an inactive enzyme, but to the amount of the enzyme. Our preliminary findings by dot blot hybridization, using a 32P-labeled cDNA probe for PNMT suggest that differences in adrenal and medulla oblongata PNMT activity between Fischer and Buffalo rats are partially due to differences in the amount of PNMT mRNA present.

Adrenal Glands↗