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M Sander

Publications and source records attributed to M Sander.

At least 73 records · Page 4Linked to original sources
PubMed1994

Role of adrenal renin in the regulation of adrenal steroidogenesis by corticotropin.

The major regulator of mineralocorticoid production in the adrenal is angiotensin II produced by the action of renal renin. The discovery that the rodent adrenal also synthesizes renin and angiotensinogen suggests there is autocrine regulation of mineralocorticoid synthesis. The transgenic rat [TGR(mREN2)27] expresses the Ren-2d gene predominantly in the adrenal. Despite suppressed kidney and plasma renin, these animals develop fulminant hypertension between 5 and 15 weeks of age. Corticosteroid concentrations are significantly elevated during hypertension development. We assessed steroidogenesis in TGR(mREN2)27 rats by analyzing the expression of the mRNAs for three steroidogenic enzymes: P450scc, the rate-limiting step of steroidogenesis; P450c11 beta, which converts deoxycorticosterone to corticosterone in the zona fasciculata/reticularis; and P450c11AS, which converts deoxycorticosterone to aldosterone in the zona glomerulosa. P450c11AS mRNA, but neither P450c11 beta nor P450scc mRNA, was overexpressed in the adrenal gland of TGR(mREN2)27 rats. In situ hybridization with specific probes for P450c11 beta and P450c11AS mRNA localized the former exclusively to the zona fasciculata and the latter to the zona glomerulosa. In TGR(mREN2)27 rats, the size of the adrenal and number of P450c11AS-expressing zona glomerulosa cells were about twice those of a normal Sprague-Dawley rat. Both animals respond to corticotropin similarly; corticotropin had no effect on the expression of P450scc and P45011 beta mRNAs, rendered P450c11AS mRNA undetectable, and simultaneously altered the morphology of the adrenal cortex, resulting in a lack of zona glomerulosa-like cells. Thus, the local renin-angiotensin system has a major effect on the basal expression of P450c11AS mRNA, but little effect on the corticotropin-regulated expression of P450scc, P450c11 beta, and P450c11AS mRNAs.

Adrenal Glands↗
PubMed1993

Characterization of the apurinic endonuclease activity of Drosophila Rrp1.

Drosophila Rrp1 (Recombination repair protein 1) belongs to a family of DNA repair nucleases that includes Escherichia coli exonuclease III, Streptococcus pneumoniae exonuclease A, bovine BAP, mouse APEX endonuclease, and human APE. Within a 252 amino acid region, colinear homology is shared between all members. Rrp1 is unique in that it includes a 427 amino acid N-terminal region not related to any known sequence. The protein copurifies with an apurinic endonuclease and a double-stranded DNA 3'-exonuclease. In this study, a 5'-end-labeled 37 base pair oligonucleotide substrate containing a single apurinic site was used to characterize the endonuclease activity of Rrp1. This substrate is utilized efficiently by Rrp1: the specific activity observed is 1 x 10(5) units/mg. The abasic double-stranded DNA oligonucleotide is cleaved only at the abasic site to create a single-strand break. Strand breaks are not detected in the complementary strand, in the single-stranded DNA oligonucleotide, or in the base-paired control substrate. After endonucleolytic cleavage at the abasic site, exonucleolytic processing at the nick is slow and requires a molar excess of Rrp1, while exonuclease III degrades the nicked substrate more efficiently. The Rrp1 cleavage product comigrates with a DNaseI cleavage product, and the newly formed terminus supports DNA synthesis by DNA polymerase. Therefore, Rrp1 cleaves the phosphodiester backbone at one position 5' to the apurinic site and leaves a 3'-hydroxyl terminus. Rrp1 is a class II apurinic endonuclease and is likely to be important in DNA repair in Drosophila.

Animals↗
PubMed1993

Drosophila Rrp1 complements E. coli xth nfo mutants: protection against both oxidative and alkylation-induced DNA damage.

Drosophila Rrp1 protein has four tightly associated enzymatic activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease and apurinic/apyrimidinic (AP) endonuclease. The carboxy-terminal region of Rrp1 is homologous to Escherichia coli exonuclease III and several eukaryotic AP endonucleases. All members of this protein family cleave abasic sites. Rrp1 protein was expressed under the control of the E. coli RNA polymerase tac promoter (pRrp1-tac) in two repair deficient E. coli strains (BW528 and LG101) lacking both exonuclease III (xth) and endonuclease IV (nfo). Rrp1 confers resistance to killing by oxidative, antitumor and alkylating agents that damage DNA (hydrogen peroxide, t-butylhydroperoxide, bleomycin, methyl methanesulfonate, and mitomycin C). Complementation of the repair deficiency by Rrp1 provides up to a two log increase in survival and requires the C-terminal nuclease region of Rrp1, but not its N-terminal region. The AP endonuclease activity in extracts from the repair deficient strain LG101 is increased up to 12-fold when the strain contains pRrp1-tac. These results indicate that pRrp1-tac directs the synthesis of active enzyme, and that the nuclease activities of Rrp1 are likely to be the cause of the increased resistance to DNA damage of the mutant cells.

Alkylation↗
PubMed1993

Expression of Drosophila Rrp1 protein in Escherichia coli. Enzymatic and physical characterization of the intact protein and a carboxyl-terminally deleted exonuclease-deficient mutant.

Drosophila Rrp1 protein purified from embryos has four tightly associated enzymatic activities: DNA strand transfer, single-strand DNA renaturation, 3'-exonuclease, and apurinic endonuclease. Copurifying with these activities is a single polypeptide that has an apparent M(r) of 105,000 when estimated by SDS-polyacrylamide gel electrophoresis. To determine if this polypeptide is sufficient for these activities, it has been overexpressed in Escherichia coli. In crude extracts of E. coli cells, an ATP-independent Mg(2+)-dependent strand transfer activity is observed upon activation of the promoter that drives expression of Rrp1. Rrp1 protein purified from induced E. coli cells has electrophoretic, chromatographic, and enzymatic properties similar to those of Drosophila Rrp1 protein. The carboxyl-terminal region of Rrp1 (amino acids 428-679) is homologous to E. coli exonuclease III. Rrp1 deleted for this region cannot carry out DNA strand transfer, but can renature complementary single-strand DNA. The strand transfer activity of this truncated protein can be restored if DNA 3'-exonuclease is provided in trans by pretreating the double-strand DNA substrate with E. coli exonuclease III. This demonstrates a likely role of the exonuclease in the in vitro DNA strand transfer reaction carried out by Rrp1 protein. Such a role is also suggested by an analysis of the polarity of the strand transfer reaction.

Animals↗
PubMed1992

Role of tissue renin in the pathophysiology of hypertension in TGR(mREN2)27 rats.

A transgenic rat line, TGR(mREN2)27, was established by introducing the murine Ren-2 gene into the genome of rats by microinjection techniques. These rats exhibit severe hypertension, making them an interesting model in which to study the role of renin in the pathophysiology of hypertension. However, although the additional renin gene is the only genetic difference compared with control rats, the exact mechanism of hypertension in TGR(mREN2)27 rats is still unclear. It cannot be attributed to a stimulation of the endocrine renin-angiotensin system or to an overexpression of renin in the kidney, since plasma and kidney renin and renin gene expression in the kidney are low in these animals. Here we describe recent progress made toward elucidating mechanisms of hypertension in TGR(mREN2)27 rats. 1) TGR(mREN2)27 rats were bred to homozygosity. The development of high blood pressure in homozygous rats is accelerated compared with that of heterozygous rats. This is paralleled by a higher mortality rate in homozygous TGR(mREN2)27 rats. Blood pressure and mortality rate of homozygous transgenic rats were effectively reduced by 10 mg captopril per kilogram body weight. 2) Treatment of 8-week-old heterozygous TGR(mREN2)27 rats with 10 mg/kg body wt per day of the angiotensin II receptor antagonist DuP 753 for 4.5 weeks normalized blood pressure. After withdrawal of the drug, blood pressure increased rapidly, reaching control levels after 3 weeks. In another group of TGR(mREN2)27 rats treated with 0.5 mg/kg per day, there was no change in blood pressure. Plasma renin and plasma angiotensin II were significantly higher in the high-dose group compared with the low-dose group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Hormones↗
PubMed1992

The role of the adrenal gland in hypertensive transgenic rat TGR(mREN2)27.

The TGR(mREN2)27 is a new monogenetic rat model in hypertension research. As the mouse Ren-2d renin gene is integrated into their genome, they develop fulminant hypertension between 5 and 15 weeks of age, with blood pressure maxima of 300 mm Hg. Their plasma renin-angiotensin system (RAS) is suppressed, but the transgene is highly expressed in the adrenal gland, so we investigated its possible role in steroid metabolism and the pathogenesis of hypertension. During the phase of hypertension development (between 6-18 weeks), the urinary excretion of deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone, and aldosterone is 1.5- to 2.5-fold elevated compared with that in Sprague-Dawley (SD) rats (P less than 0.0005) despite the suppressed plasma RAS. Moreover, the adrenal gland in TGR(mREN2)27 shows an increased maximal response to ACTH stimulation in regard to urinary excretion of DOC (after ACTH, 244 +/- 42 ng/24 h in TGR; 62 +/- 10 ng/24 h in SD; P less than 0.0005) and B (after ACTH, 5144 +/- 346 ng/24 h in TGR; 2607 +/- 324 ng/24 h in SD; P less than 0.0005). Additionally, plasma prorenin in TGR was stimulated more than 10-fold, indicating transgene regulation by ACTH. Since spironolactone treatment did not lower the blood pressure in TGR, hypertension solely due to hypermineralocorticoism is unlikely. Our results indicate that the adrenal steroid metabolism is markedly stimulated in young TGR, and the absolute increase in urinary DOC and B after ACTH injections is enhanced, possibly due to a stimulated local intraadrenal RAS.

18-Hydroxycorticosterone↗
PubMed1991

Cloning and characterization of Rrp1, the gene encoding Drosophila strand transferase: carboxy-terminal homology to DNA repair endo/exonucleases.

We previously reported the purification of a protein from Drosophila embryo extracts that carries out the strand transfer step in homologous recombination (Lowenhaupt, K., Sander, M., Hauser, C. and A. Rich, 1989, J. Biol. Chem. 264, 20568). We report here the isolation of the gene encoding this protein. Partial amino acid sequence from a tryptic digest of gel purified strand transfer protein was used to design a pair of degenerate oligonucleotide primers which amplified a 635 bp region of Drosophila genomic DNA. Recombinant bacteriophage were isolated from genomic and embryo cDNA libraries by screening with the amplified DNA fragment. These bacteriophage clones identify a single copy gene that expresses a single mRNA transcript in early embryos and in embryo-derived tissue culture cells. The cDNA nucleotide sequence contains an open reading frame of 679 amino acids within which are found 5 tryptic peptides from the strand transfer protein. Expression of this cDNA in E. coli produces a polypeptide with the same electrophoretic mobility as the purified protein. The deduced protein sequence has two distinct regions. The first 427 residues are basic, rich in glutamic acid and lysine residues and unrelated to known proteins. The carboxy-terminal 252 residues are average in amino acid composition and are homologous to the DNA repair proteins, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A. This protein, which we name Rrp1 (Recombination Repair Protein 1), may facilitate recombinational repair of DNA damage.

Amino Acid Sequence↗
PubMed1991

Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities.

A protein previously purified from Drosophila embryo extracts by a DNA strand transfer assay, Rrp1 (recombination repair protein 1), has an N-terminal 427-amino acid region unrelated to known proteins, and a 252-amino acid C-terminal region with sequence homology to two DNA repair nucleases, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A, which are known to be active as apurinic endonucleases and as double-stranded DNA 3' exonucleases. We demonstrate here that purified Rrp1 has apurinic endonuclease and double-stranded DNA 3' exonuclease, activities and carries out single-stranded DNA renaturation in a Mg(2+)-dependent manner. Strand transfer, 3' exonuclease, and single-stranded DNA renaturation activities comigrate during column chromatography. The properties of Rrp1 suggest that it could promote homologous recombination at sites of DNA damage.

Amino Acid Sequence↗
PubMed1989

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites.

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.

Animals↗
PubMed1989

Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein.

The purification of a Drosophila strand transfer protein is described, which involves Bio-Rex 70, Superose 6, Mono S, and single-stranded DNA-agarose chromatography. A 105,000-dalton polypeptide copurifies with the strand transfer activity on the last two column steps. The strand transferase carries out strand transfer at an unusually low protein:single-stranded DNA ratio and requires neither a nucleotide cofactor nor exogenous single-strand DNA binding protein to form heteroduplex DNA. Biochemical analysis of the reaction products has established that one strand of the DNA duplex is displaced during the reaction. Several properties, including the kinetics and stoichiometry of strand transfer, differentiate this activity from previously characterized strand transferases.

Adenosine Triphosphate↗
PubMed1989

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II.

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.

Animals↗
PubMed1989

[Results of physiotherapy for patients with myofacial dysfunction].

The methods and results of physiotherapy in the treatment of 200 patients with myofacial pain are described. The first step in this treatment consists of exercises to enhance body awareness. This is followed by mobilization of the entire locomotor system and manual massages for muscle relaxation. Subsequently coordinated physical exercises are performed to train complex movement patterns of masticatory, facial and trunk muscles. 127 patients (64%) could be cured by means of physiotherapy and behavioral guidance alone, another 53 patients (27%) with additional bite plane therapy.

Adult↗
PubMed1988

Influence of marathon running on thyroid hormones.

Thyroid hormones were studied in 16 well-trained male amateur runners (mean age 31.8 years) before, immediately after, and 60 min and 22 h after marathon running. Free thyroxine (fT4), appraised by the free thyroxine index (fT4-I), was significantly increased immediately after and 1 h after the race compared with control values. Thyroid-stimulating hormone (TSH) was significantly increased immediately after the race, returned to the control value 1 h later, and was markedly decreased 22 h after the race. Free triiodothyronine (fT3) and free reverse triiodo-thyronine (frT3), appraised by the ratios triiodothyronine/-thyroxine binding globulin and reverse triiodothyronine/thyroxine binding globulin, respectively, showed contrary results. frT3 increased significantly after exercise, whereas fT3 decreased insignificantly. The peripheral conversion of thyroxine was additionally estimated by the ratios rT3/T3 and rT3/T. Both ratios were increased significantly after the run and remained elevated 22 h after the race. It is concluded that an increased TSH-regulated T4 secretion occurs during prolonged exercise as well as a change of the peripheral conversion process in favor of the hormonal inactive rT3. The latter condition remained still 22 h after the race.

Adolescent↗
PubMed1988

Sensitive homologous recombination strand-transfer assay: partial purification of a Drosophila melanogaster enzyme and detection of sequence effects on the strand-transfer activity of RecA protein.

A sensitive homologous recombination strand-transfer assay is described that employs short radiolabeled double-stranded DNA fragments from the lac/polylinker region of plasmid pUC18 and (+)viral M13mp18 single-stranded DNA as substrates. Substitution of a short radiolabeled double-stranded fragment for full-length linear M13 double-stranded DNA results in an assay whose sensitivity is improved greater than 8-fold. In addition, it is less sensitive to interference from nucleases or ligases than previous assays. The assay was used to partially purify an ATP-independent strand-transfer activity from a crude nuclear extract of Drosophila melanogaster embryos. We have also tested the efficiency with which various short double-stranded DNA segments are assembled into plectonemic joints by RecA protein with this assay and found 5- to 10-fold differences. These results are interpreted as evidence for DNA sequence-specific effects in RecA-mediated homologous pairing in vitro.

Animals↗
PubMed1987

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding.

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.

Animals↗