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R S Hansen

Publications and source records attributed to R S Hansen.

At least 37 records · Page 2Linked to original sources
PubMed1993

Association of fragile X syndrome with delayed replication of the FMR1 gene.

The fragile X syndrome is commonly associated with mutant alleles of the FMR1 gene that are hypermethylated and have large expansions of CGG repeats. We present data here on the replication timing of FMR1 that confirm predictions of delayed replication of alleles from affected males. The normal FMR1 allele replicates late in S phase, while alleles from affected males replicate later, the major peak of replication occurring in the flow cytometry fraction usually referred to as G2/M. The delayed timing of replication is not the direct result of a single replication fork stalling at the expanded CGG repeat, because delayed replication was observed for regions on both sides of the repeat. The domain of altered replication timing includes sites at least 150 kb 5' and 34 kb 3' of the repeat, indicating that genes in addition to FMR1 may be affected.

Animals↗
PubMed1992

Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid.

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.

Animals↗
PubMed1990

5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region.

5-Methylcytosine↗
PubMed1990

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

The 5' region of the gene encoding human X chromosome-linked phosphoglycerate kinase 1 (PGK1) is a promoter-containing CpG island known to be methylated at 119 of 121 CpG dinucleotides in a 450-base-pair region on the inactive human X chromosome in the hamster-human cell line X8-6T2. Here we report the use of polymerase chain reaction-aided genomic sequencing to determine the complete methylation pattern of this region in clones derived from X8-6T2 cells after treatment with the methylation inhibitor 5-azacytidine. We find (i) a clone showing full expression of human phosphoglycerate kinase is fully unmethylated in this region; (ii) clones not expressing human phosphoglycerate kinase remain methylated at approximately 50% of CpG sites, with a pattern of interspersed methylated (M) and unmethylated (U) sites different for each clone; (iii) singles, defined as M-U-M or U-M-U, are common; and (iv) a few CpG sites are partially methylated. The data are interpreted according to a model of multiple, autonomous CpG sites, and estimates are made for two key parameters, maintenance efficiency (Em approximately 99.9% per site per generation) and de novo methylation efficiency (Ed approximately 5%). These parameter values and the hypothesis that several independent sites must be unmethylated for transcription can explain the stable maintenance of X chromosome inactivation. We also consider how the active region is kept free of methylation and suggest that transcription inhibits methylation by decreasing Em so that methylation cannot be maintained. Thus, multiple CpG sites, independent with respect to a dynamic methylation system, can stabilize two alternative states of methylation and transcription.

Animals↗
PubMed1988

Purification of calmodulin-stimulated cyclic nucleotide phosphodiesterase by monoclonal antibody affinity chromatography.

Immobilized ACC-1 and ACAP-1 antibodies are effective tools for the purification of active calmodulin-dependent phosphodiesterases. ACC-1 antibody binds all bovine and rat brain isozymes in a Ca2+-dependent manner and has been used for their purification. Since ACC-1 binds both bovine brain isozymes (61- and 63-kDa forms) and ACAP-1 recognizes only the 61-kDa isozyme, ACAP-1 can be used to separate and purify the two brain isozymes. The procedures described here for phosphodiesterase isolation from brain are rapid and require few enzymatic assays, resulting in preparations of good purity, specific activity, and yield (Tables II, III). The procedures for brain tissue can be easily adapted for use with larger amounts of tissue. The cross-reactivity of ACP-1 for rat brain phosphodiesterase suggests that this antibody may recognize isozymes from other mammalian tissues.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression.

X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.

Animals↗
PubMed1986

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody.

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity.

Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.

Amino Acid Sequence↗
PubMed1985

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.

Animals↗
PubMed1985

The acquired immunodeficiency syndrome.

AIDS is an apparently new condition that first occurred in about 1979 and is manifested primarily by profound disturbances of T-cell immunity and unusual susceptibility to either opportunistic infections (mycobacterial, fungal, parasitic, or viral) or tumors such as Kaposi's sarcoma and lymphoma. A prodrome of lymphadenopathy and wasting is also part of the spectrum of disease. The etiology is unknown, but the likely candidates are viral agents. Epidemiologic studies show that the promiscuous gay male is at particular risk, but transmission by the parenteral route and/or vertical transmission has extended the risk of acquisition to other groups. A common clinical presentation of full-blown AIDS is opportunistic infection of the lungs (especially with P. carinii), the central nervous system, or the gastrointestinal tract. Therapy against specific pathogens may be temporarily successful, but the course is frequently downhill, with relentless progressive or sequential infections or development of a tumor during continued immunodepression. Therapy for the immunologic abnormalities remains experimental. Control measures to prevent transmission are similar to those for hepatitis B.

Acquired Immunodeficiency Syndrome↗
PubMed1984

2,4-Diamino-5-cyano-6-halopyridines: a new class of cyclic AMP phosphodiesterase inhibitors with therapeutic potential.

2,4-Diamino-5-cyano-6-halopyridines have been described previously as oral insulinotropic agents and have been found recently to have bronchodilatory properties. In the present report the synthesis of the iodo compound is newly described, and it is established that HI- or HBr-mediated condensation and cyclization of malononitrile in 1,2-dichloroethane yield selectively the 5-cyanopyridine derivatives. The pyridine derivatives were found to constitute a new class of potent cyclic AMP phosphodiesterase inhibitors. Inhibition of purified dog kidney cyclic AMP phosphodiesterase was of the mixed type. Since cyclic AMP phosphodiesterase inhibitors are known to enhance glucose-induced insulin secretion and to activate glucose production by the liver, the finding that the pyridine derivatives described here inhibited cyclic AMP phosphodiesterase opens new avenues of interpretation for their insulinotropic action as well as for the paradoxical lack of improvement of glucose disposal by elevation of insulin after oral drug administration. Cyclic AMP phosphodiesterase inhibition also has the potential of explaining the bronchodilatory effects of these drugs.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Immunologic approaches to the study of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide PDE activity in crude tissue extracts is due to the composite activity of several distinct isozymes, each having different kinetic and functional characteristics. Most of these isozymes will hydrolyze the same substrates, that is, cyclic AMP and cyclic GMP. Therefore, it is difficult to conclusively identify and quantitate any given isozyme in crude systems. Since the cyclic nucleotide PDE are present in low concentrations in cells, it is also difficult to isolate these proteins without having specific solid phase affinity probes. This chapter described the use of monoclonal antibodies as probes to specifically identify, measure, characterize, and isolate the PDE isozymes in impure preparations. Monoclonal antibodies have been produced to the cyclic-GMP-stimulated PDE, ROS PDE, and a calcium-calmodulin-dependent PDE of bovine tissues. A polyclonal antiserum has also been produced to the cyclic-GMP-stimulated PDE. Sensitive immunoprecipitation assays for the measurement of PDE activity and cyclic GMP binding have been developed that have allowed the detection of femtomole amounts of each specific PDE isozyme. None of the antibodies appear to recognize antigenic determinants on other isozymes, suggesting that the PDE are antigenically distinct and therefore different proteins. Most of the monoclonal antibodies do not appear to affect kinetic parameters of the enzymes and have been used to specifically identify, measure, and characterize each isozyme in crude systems. A monoclonal antibody to the calcium-calmodulin-dependent PDE requires calcium and calmodulin for high-affinity binding to the enzyme. This apparent conformational requirement has been used to purify the isozyme from both bovine brain and heart. These two tissues contain enzymes with differing subunit MW on SDS-polyacrylamide gel electrophoresis, although no other biochemical differences were observed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1982

Purification of two calcium/calmodulin-dependent forms of cyclic nucleotide phosphodiesterase by using conformation-specific monoclonal antibody chromatography.

A procedure for nondenaturing immunopurification of bovine calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) is described that utilizes chromatography on a conformation-specific monoclonal antibody column. Hybridomas derived from spleen cells of mice immunized with Ca(2+)/calmodulin/phosphodiesterase were screened for antiphosphodiesterase antibody production. A stable cell line was established that secretes a monoclonal antibody that binds to the Ca(2+)/calmodulin/enzyme complex with an approximate K(d) of 10(-9) M. The dissociation constant was increased by two orders of magnitude when calmodulin interaction with the enzyme was inhibited by Ca(2+) chelation. This differential reactivity was utilized for affinity chromatography of heart and brain phosphodiesterases on monoclonal antibody columns. Highly purified phosphodiesterases were eluted in good yield with buffer containing EGTA. The immunopurified enzymes from heart and brain exhibited specific activities of approximately 300 units/mg when assayed at millimolar concentrations of cGMP or cAMP. Calmodulin stimulated both enzymes 10- to 15-fold over basal activity under these conditions. However, analysis of the two preparations by NaDodSO(4)/polyacrylamide gel electrophoresis revealed an apparent subunit of M(r) 61,000 for the brain enzyme, in contrast to the M(r) 59,000 cardiac subunit. The observed difference was not an artifact of tissue homogenization because both forms were detected after purification from mixed-tissue homogenates. These results suggest that mild, biospecific elution from a conformation-specific monoclonal antibody column may be a general technique applicable to the rapid isolation of proteins whose antigenic determinants can be altered with specific ligands.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Nervousness and hysteria of mature female chickens.

A type of abnormal nervousness and hysteria, e.g., adding nests and perches to community cages and and hysteria affecting White Leghorn female chickens 35 or more weeks of age was investigated in nine experiments. Experimental flocks in which the malady consistently occurred were housed in large group (community) cages. Limited information was obtained on three cases in commercial flocks housed on the floor. Social pressure resulting from high population density appeared to be primary causative factor, with pain apparently contributing to the final break to hysteria. Claw removal at one day of age prevented hysteria but not nervousness in one experiment. Colored light, feeding added niacin or a mild tranquilizer were not successful preventives. Short term feeding of a high level of niacin, claw trimming and forced molting afforded relief in some cases while a heavy sedative was ineffective as a curative. Modification of the environment to reduce social pressure was successful in preventing nervousness and hysteria, e.g., adding nests and perches to community cages and moving flocks to less crowded housing. Strain differences in tendency to develop hysteria were demonstrated. There were positive indications of physiological changes associated with hysteria.

Animals↗