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Biomedical subjects

D Werner

Publications and source records attributed to D Werner.

At least 109 records · Page 6Linked to original sources
PubMed1990

Urate and p-aminohippurate transport in the brush border membrane of the pig kidney.

The transport of urate and p-aminohippurate (PAH) across the pig renal brush border membrane was investigated using membrane vesicles. Compared to a pH equilibrium condition (pHin = 7.4; pHout = 7.4), an outwardly directed OH- gradient (pHin = 7.4; pHout = 5.8) stimulated markedly both lactate and pyrazinoate uptake with overshoots exceeding 2 times that of the steady state. In contrast, neither OH-/urate nor OH-/PAH exchange could be demonstrated. An outwardly directed Cl- gradient (Cl-in = 100 mM; Cl-out = 16.7 mM) increased 2.6-fold 15-sec PAH uptake compared to Cl- equilibrium (Cl-in = Cl-out = 100 mM) but this stimulation was due solely to an effect of membrane potential. Creation of an electropositive intravesicular space, by imposing an inwardly directed K+ gradient (K+in = 0 mM; K+out = 100 mM) in the presence of valinomycin, was very effective to drive uphill PAH and urate accumulation compared to the control condition (no valinomycin). Four-second potential-stimulated PAH uptake was saturable (Km, 0.8 mM; Vmax, 1.7 nmol/mg of protein x 4 sec). Different organic anions cis-inhibited both 4-sec potential-stimulated PAH and urate uptakes in a similar fashion. PAH and urate, moreover, decreased each other's accumulation. In countertransport experiments, PAH and urate uptakes were not significantly stimulated by trans-unlabeled substrate. These results are consistent with the presence, in the pig renal brush border membrane, of a mediated secretory pathway common for urate and PAH, which could be facilitated by the potential difference existing in vivo across the luminal membrane of the proximal tubule.

Aminohippuric Acids↗
PubMed1989

Site-specific location of covalent DNA-polypeptide complexes in the chicken genome.

A nitrocellulose filter binding assay was applied to isolate and to analyze the fraction of chicken DNA fragments associated with residual nuclear polypeptides resistant to SDS/proteinase K treatment and phenol extraction. It is shown that the DNA-polypeptide complexes retained on nitrocellulose filters are located on a non-random sub-set of DNA sequences. (a) Southern analysis reveals that the fractions of DNA fragments from chicken erythrocytes and from hen oviduct cells associated with the resistant polypeptides have a lower sequence complexity than unfractionated DNA. Moreover, the retained DNA fractions from different cell types of the same species are highly homologous. (b) All DNA fragments of the transcriptionally active and inactive ovalbumin gene map in the DNA fraction passing the filters indicating that the tight DNA-polypeptide complexes are not remnants of transcription complexes. (c) By use of a genomic sub-set library prepared from DNA retained on filters, clones were isolated with sequences mapping specifically in the DNA fraction associated with the tight DNA-polypeptide complexes. The results are consistent with fixed covalent DNA-polypeptide complexes in the chicken genome whose location is essentially identical in different cell types of the same species and apparently determined by DNA signal-sequences.

Animals↗
PubMed1989

Nucleotide sequence of the full-length mouse lamin C cDNA and its deduced amino-acid sequence.

We have cloned and sequenced the cDNA comprizing the entire coding region and several hundred base-pairs of its flanks for the mouse nuclear envelope protein lamin C mRNA. The nucleotide sequence and the deduced amino-acid sequence of the mouse lamin C are compared with the previously published human lamin A/C sequences with respect to (a) the general organisation, (b) homologies, (c) predictions for the essential structural characteristics of lamins and (d) the localization of the most conserved region. Moreover, the mouse lamin C sequence presented allows the first intraspecies comparison between A/C-type and B-type lamins.

Amino Acid Sequence↗
PubMed1988

Construction and quality of cDNA libraries prepared from cytoplasmic RNA not enriched in poly(A)+RNA.

Poly(A)+RNA and cytoplasmic RNA of Ehrlich ascites tumor cells grown in vivo were used to study the quality and efficiency of cDNA synthesis. It was found that the rates of oligo(dT)-primed and unprimed reverse transcription were very similar in both cases. The size distributions of the cDNA strands prepared from unfractionated RNA reflected the size of cytoplasmic mRNA populations including a significant fraction of long molecules up to 6 kb. The fraction of cDNAs primed on rRNAs by oligo(dT) was found to be as low as 2-3%. Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template. In repeated experiments we obtained an average yield of 2.2 micrograms of double-stranded cDNA when 70 micrograms of unfractionated cytoplasmic RNA was used as starting material. This amount of cDNA synthesized in one assay was sufficient to construct representative cDNA libraries in different vectors. Southern hybridizations of DNA isolated from cDNA libraries with various radiolabelled probes show that the libraries constructed from cDNA synthesized from cytoplasmic RNA not enriched in poly(A)+RNA contain a high ratio of full-length cDNA clones. The results suggest that representative cDNA libraries of high quality can be constructed without pre-isolation of poly(A)+RNA fractions.

Animals↗
PubMed1988

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

Animals↗
PubMed1988

Carnitine plasma concentrations in 353 metabolically healthy children.

Carnitine plasma concentrations were determined by an enzymatic radioisotopic method in 353 metabolically healthy children and in 41 adults. There was a positive correlation between total and free carnitine plasma concentrations and the age of the children. Both free and acylcarnitine concentrations were elevated on the 1st day of life, reflecting an increased rate of fatty acid oxidation. Carnitine plasma concentrations decreased after the 1st day and subsequently increased during the 1st year. From the 2nd year of life until adulthood, no further change was noted. Up to 17 years of age no differences were seen between male and female individuals. However, adult males had higher carnitine concentrations in plasma than adult females. Total carnitine concentrations were higher in 10- to 17-year-old females and lower in 10- to 17-year-old males compared with adults of the same sex, indicating a possible role for sex hormones in the regulation of carnitine plasma concentrations.

Adolescent↗
PubMed1988

Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo.

Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination. Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl-. EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53. The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries. Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase. The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells. Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries. No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications. Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest. Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.

Animals↗
PubMed1988

The role of U tube palliative treatment in high bile duct carcinoma.

Twenty-one patients with cholangiocarcinoma at the confluence of the main right and left hepatic ducts were referred to our professorial surgical unit between 1968 and 1982. All were evaluated, treated, and documented prospectively with follow-up to mid 1986. No lesion was deemed resectable. The U tube palliative bypass developed during the course of the study was used in 14 patients, and its role in treating high bile duct carcinoma was evaluated. Histologic confirmation of the diagnosis was obtained in 71% of patients. Seven patients received additional treatment with radical radiotherapy. The 30-day overall hospital mortality rate was 19%. The 1- and 2-year survival rates were 57% and 33%, respectively. The quality of survival was usually good. The need for centralized referral and treatment of these difficult patients is stressed. The case against radical resection for this lesion is presented. It is concluded that radical resection is seldom possible, and therefore the U tube palliative procedure is advocated in most patients.

Adenoma, Bile Duct↗
PubMed1987

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA.

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.

Animals↗
PubMed1987

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation.

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.

Cell Nucleus↗
PubMed1986

Amino-acid-transfer reactions in isolated nuclei of Ehrlich ascites tumor cells.

Nuclear enzymatic activities incorporating amino acids into acid-insoluble material were investigated with respect to their differentiation from protein biosynthesis, reaction optima, requisites and localization. The product of the reaction was analyzed with respect to its localization and nature. The nuclear activities are not inhibited by a number of inhibitors for protein biosynthesis. The reaction optima found are similar to those of other residual nuclear syntheses including the stringent dependence on ATP. All naturally occurring amino acids are utilized with different efficiencies. Their incorporation is neither cooperative nor competitive which points to individual incorporation mechanisms. Aminoacylation of tRNA may be involved because the incorporation is RNase-sensitive and aminoacylation of tRNA can be shown under the reaction conditions. The enzymatic activities are exclusively nuclear. Significant activity with unchanged characteristics is released by sonication. 70% of the radiolabel incorporated is exported across the nuclear envelope during the incubation. The residual 30% of the radiolabel is distributed without enrichment in any nuclear subfraction. The products are exclusively of polypeptide nature. Since distinct nuclear proteins (e.g. histones) which are definitely preformed in the cytoplasm by protein biosynthesis become radiolabelled by the incorporation of radiolabelled amino acids, it is evident that the incorporation takes place at preformed polypeptides. This is unequivocally proven by the incorporation of radiolabelled amino acids into exogenous proteins by means of the solubilized nuclear activities. The results indicate that the nuclear activity under investigation reflects a nuclear modification system for polypeptides which may be of similar importance as other post-translational modification systems.

Amino Acids↗
PubMed1986

Inhibition of Lewis lung tumor growth and metastasis by Ehrlich ascites tumor growing in the same host.

A double tumor model (3LL and EAT) was established in C57BL/6 mice. The primary 3LL tumor was growth-inhibited by EAT inoculated into the same host. Growth inhibition of the 3LL primary tumor depended on the number of EAT cells injected. Inhibition of the growth of the primary 3LL tumor was also observed after injection of the optimal number (10(7)) of EAT cells 24 h to 7 days before or after inoculation of 3LL cells. In contrast, metastasis of the 3LL tumor was only inhibited after simultaneous inoculation of 3LL and ETA cells. Moreover, EAT cells inhibited the growth of preformed 3LL-derived metastases after resection of the primary 3LL tumor. Although the EAT-induced stimulation of a host defense system is not excluded, the results are consisted with the continued release of growth inhibition factors from EAT cells acting on 3LL cells in the primary tumor and in lung metastases.

Animals↗
PubMed1986

Disintegration of nucleoskeletal elements by metrizamide/2 M salt isopyknic centrifugation.

Supramolecular structures that remain bound to chromosomal DNA under high salt conditions are believed to anchor DNA in the interphase nuclear skeleton. In order to identify these anchorage structures, the non-DNA materials that remain firmly bound to chromosomal DNA under conditions that disintegrate the high salt-stable architecture of nuclei were investigated. Nuclei of Ehrlich ascites cells were histone-depleted by treatment with 2 M salt. The residual halo structures were gently sheared and subjected to metrizamide isopyknic centrifugation in the presence of 2 M salt. By this combined treatment the high salt stable nuclear skeleton becomes disintegrated and three main fractions are resolved. A light fraction comprises the DNA which appears to be essentially depleted of other nuclear components. The only non-DNA material that could be identified in the DNA band is a fraction of (nascent) RNP. No other materials which could reflect nucleoskeletal elements (e.g. lamina proteins) were found together with DNA. A peak of intermediate density comprises RNA/RNP dissociated from DNA. The heavy fraction contains the proteins that become dissociated from DNA by high-salt and/or centrifugal forces, e.g. histones and the major nuclear lamina proteins. The results indicate that nascent RNP is more tightly bound to chromosomal DNA than other components that may be involved in nuclear skeletons. This suggest that transcription complexes represent at least one type of anchorage structure of DNA, which is consistent with results indicating that nascent RNA and actively transcribed DNA sequences are preferentially retained in high-salt-treated nuclei.

Animals↗
PubMed1986

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation.

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.

Animals↗
PubMed1986

Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes.

Host-cell cytoplasm from soybean plants infected with the peribacteroid membrane (PBM)-building Rhizobium japonicum strain 61-A-101 (effective, N(2)-fixing) had much higher choline kinase activity than cytoplasm from either uninfected tissue or tissue infected with the non-PBM-building (ineffective, non-N(2)-fixing) strain 61-A-24. Ion-exchange chromatography showed that both types of nodule and root tissue possessed constitutive choline kinase I activity that had a K(m) for choline of approximately 150 muM. The nodules of the effective symbiosis had another activity, choline kinase II (K(m) = 81 muM). Nondenaturing and NaDodSO(4) electrophoresis revealed no multimeric subunit structure of the two enzyme forms but did show the molecular sizes for choline kinase I, 58-59 kDa, and choline kinase II, 60 kDa. Choline kinase I and II and pI values of 8.1 and 8.5, respectively, and two-dimensional gel electrophoresis of whole cytoplasm from control and infected tissue showed a spot corresponding to choline kinase II only in the case of the effective symbiosis, whereas both tissue types had spots corresponding to choline kinase I. Choline kinase II is presumed to be encoded by the plant as neither free-living nor symbiotic (bacteroid) forms of the prokaryote showed any choline kinase activity.

Journal Article↗
PubMed1986

Thiazide-induced hyponatremia: an unusual neurologic course.

We have presented a case of thiazide-induced hyponatremia that followed an unusual course after the patient, suffering from extreme lethargy, was admitted with a serum sodium concentration of 104 mEq/L. Although the electrolyte imbalance was essentially corrected within 20 hours and there was clinical improvement, three days later the patient suddenly lost consciousness. She was comatose for more than a month, apparently from extrapontine myelinolysis. After a dramatic improvement, the patient was discharged with almost no neurologic deficit.

Acute Disease↗