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Biomedical subjects

D Werner

Publications and source records attributed to D Werner.

At least 91 records · Page 5Linked to original sources
PubMed1992

Isoliquiritigenin, a strong nod gene- and glyceollin resistance-inducing flavonoid from soybean root exudate.

Isoflavonoid signal molecules from soybean (Glycine max (L.) Merr.) seed and root exudate induce the transcription of nodulation (nod) genes in Bradyrhizobium japonicum. In this study, a new compound with symbiotic activity was isolated from soybean root exudate. The isolated 2',4',4-trihydroxychalcone (isoliquiritigenin) is characterized by its strong inducing activity for the nod genes of B. japonicum. These genes are already induced at concentrations 1 order of magnitude below those required of the previously described isoflavonoid inducers genistein and daidzein. Isoliquiritigenin is also a potent inducer of glyceollin resistance in B. japonicum, which renders this bacterium insensitive to potentially bactericidal concentrations of glyceollin, the phytoalexin of G. max. No chemotactic effect of isoliquiritigenin was observed. The highly efficient induction of nod genes and glyceollin resistance by isoliquiritigenin suggests the ecological significance of this compound, although it is not a major flavonoid constituent of the soybean root exudate in quantitative terms.

Benzopyrans↗
PubMed1992

[The incidence of silent myocardial ischemia].

Retrospectively evaluated was the patient population of the years 1982 to 1988 which underwent ergometric investigations concerning the appearance of silent myocardial ischaemias. In 256 reactions of exercise ischaemia of patients with definitive coronary disease in 47 cases angina pectoris appeared (18.4%). Reactions of ischaemia in a large area of the breast wall showed, compared with a small area of ischaemia, a significantly higher proportion of symptomatic episodes (34.3% vs 13.0%, P less than 0.05). Ergometric exercises lying below the norm of age were more frequently accompanied by symptomatic ischaemias than ischaemic reactions, which only appeared in a good area of performance (26.0% vs 11.4%, P less than 0.05). In addition to this more anginose complaints were found in patients, whose ischaemic reaction was accompanied by an insufficient frequency response (28.1% vs 9.8%, P less than 0.05).

Angina Pectoris↗
PubMed1991

Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA.

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.

Amino Acids↗
PubMed1991

Nucleotide sequence of the murine prothymosin alpha cDNA and its deduced primary and secondary protein structure.

Searching for proliferation-related and cell cycle phase-specific genes we detected a full-length cDNA for the murine prothymosin alpha mRNA which was sequenced on the DNA level. The amino acid sequence deduced from the nucleotide sequence shows a high degree of positional identities with prothymosin alpha from man and rat. However, the minor differences in the primary structures largely influence predictions for the secondary structures of prothymosin alpha from different species. These differences in the secondary structure could explain the differences of activity of prothymosin alpha from different origin in immuno-protection assays.

Amino Acid Sequence↗
PubMed1991

Circadian pattern of large neutral amino acids, glucose, insulin, and food intake in anorexia nervosa and bulimia nervosa.

Insulin, glucose, and large neutral amino acids (LNAA) were studied in 10 patients with anorexia nervosa, 13 patients with bulimia nervosa, and 15 healthy controls. Blood samples were collected at hourly intervals during the day and at two-hour intervals during the night over a 24-hour period. Ad libitum caloric and relative carbohydrate intake was significantly reduced in the anorectic and bulimic patients. Elevated concentrations of beta-hydroxybutyric acid (BHBA) were seen in the bulimic group, and low triiodothyronine (T3) concentrations in the anorectic group. Mean plasma glucose and insulin concentrations were significantly lowered in both groups. The tryptophan (Trp) to LNAA ratio was reduced in anorectic, but not in bulimic patients. These findings suggest that Trp influx into the brain is reduced in anorectic patients, possibly impairing central serotonergic function.

Amino Acids↗
PubMed1991

Chemotaxis and nod Gene Activity of Bradyrhizobium japonicum in Response to Hydroxycinnamic Acids and Isoflavonoids.

For Bradyrhizobium japonicum, the chemotactic and the nod gene-inducing effects of hydroxycinnamic acids and two of their derivatives were compared with those of isoflavonoids. Only the hydroxycinnamic acids were strong chemoattractants, while the other substances tested were chemotactically inactive. Besides the known nod gene induction by isoflavonoids, a weak nod gene induction by coniferyl alcohol, chlorogenic acid, and ferulic acid was found.

Journal Article↗
PubMed1991

Isoflavonoid-inducible resistance to the phytoalexin glyceollin in soybean rhizobia.

The antibacterial effect of the soybean phytoalexin glyceollin was assayed using a liquid microculture technique. Log-phase cells of Bradyrhizobium japonicum and Sinorhizobium fredii were sensitive to glyceollin. As revealed by growth rates and survival tests, these species were able to tolerate glyceollin after adaptation. Incubation in low concentrations of the isoflavones genistein and daidzein induced resistance to potentially bactericidal concentrations of glyceollin. This inducible resistance is not due to degradation or detoxification of the phytoalexin. The inducible resistance could be detected in B. japonicum 110spc4 and 61A101, representing the two taxonomically divergent groups of this species, as well as in S. fredii HH103, suggesting that this trait is a feature of all soybean-nodulating rhizobia. Glyceollin resistance was also inducible in a nodD1D2YABC deletion mutant of B. japonicum 110spc4, suggesting that there exists another recognition site for flavonoids besides the nodD genes identified so far. Exudate preparations from roots infected with Phytophthora megasperma f. sp. glycinea exhibited a strong bactericidal effect toward glyceollin-sensitive cells of B. japonicum. This killing effect was not solely due to glyceollin since purified glyceollin at concentrations similar to those present in exudate preparations had a much lower toxicity. However, glyceollin-resistant cells were also more resistant to exudate preparations than glyceollin-sensitive cells. Isoflavonoid-inducible resistance must therefore be ascribed an important role for survival of rhizobia in the rhizosphere of soybean roots.

Benzopyrans↗
PubMed1991

Indirect Na+ dependency of urate and p-aminohippurate transport in pig basolateral membrane vesicles.

Membrane vesicles were used to study the basolateral transport of urate and p-aminohippurate (PAH) in the proximal tubule of the pig kidney. Consistent with a cooperation between a Na(+)-2-oxoglutarate cotransporter and a 2-oxoglutarate-urate or a 2-oxoglutarate-PAH exchanger, urate and PAH uptakes were stimulated in presence of extravesicular 2-oxoglutarate when an inwardly directed Na+ gradient was applied. Both transports exhibited, however, different characteristics. The optimal 2-oxoglutarate concentration for stimulating uptakes was 10 microM for PAH and 150 microM for urate. Extravesicular chloride was required to observe a stimulation of PAH uptake but not of urate uptake. Transports of both PAH and urate exhibited different affinity sequences for various organic anions. Stimulated PAH uptake was inhibited by probenecid greater than cold PAH greater than urate = pyrazinoate greater than lactate, whereas stimulated urate uptake was inhibited by probenecid greater than cold urate greater than PAH and not by pyrazinoate or lactate. These results are consistent with independent transport processes for urate and PAH in pig basolateral membrane vesicles, both being indirectly driven by an inwardly directed Na+ gradient.

Animals↗
PubMed1991

Murine cDNAs coding for the centrosomal antigen centrosomin A.

Screening of an induced Ehrlich ascites cell-derived lambda gt11 cDNA library with an antibody (GP1), immunoreacting specifically with centrosomal antigen(s) of interphase and mitotic cells of different species, released a partial cDNA clone (lambda P10A) encoding the carboxy-terminal section of a centrosome-specific antigen. This specificity of the clone lambda P10A could be verified by lacZ-directed antigen expression from Escherichia coli Y1089 lysogenized with the recombinant phage lambda P10A and subsequent production of centrosome-specific antibodies by means of the recombinant antigen. Using the lambda P10A insert as a probe, two types of cDNA clones were identified in a lambda gt10 cDNA library by plaque-hybridization. The inserts of PN1 type clones were 1.2 kb (kilobases) and those of PN5 type clones were 2.2 kb in length. The DNA sequence of a PN1 type clone revealed its full-length cDNA nature. The open reading frame of PN1 encodes a rather hydrophilic and highly charged 34.5 x 10(3) Mr polypeptide comprising short but apparently significant strings of 100% sequence identity with the major nuclear lamina polypeptides lamins A/C and lamin B. Restriction enzyme mapping of PN1 and PN5 inserts, cross-hybridization experiments and comparison of overlapping DNA sequences indicate that the 1.2 kb and 2.2 kb cDNAs code for the same 34.5 x 10(3) Mr polypeptide, termed centrosomin A. Western blots of Ehrlich ascites cell proteins show a second, larger GP1 antigen (centrosomin B) whose cDNA has not been cloned. It remains to be investigated whether centrosomin B is encoded by a second mRNA or whether it reflects an oligomeric or a postranslationally modified form of centrosomin A.

Amino Acid Sequence↗
PubMed1990

Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli.

The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of mammalian glutathione reductase cDNA.

The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full-length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated lambda GRM-B11 was isolated by plaque-screening of an induced mouse cDNA expression library in the lambda gt11 vector with a rabbit antibody probe to human glutathione reductase. 125Iodine-labelled whole anti-rabbit immunoglobulin was used as second antibody. EcoRI digestion of the lambda GRM-B11 clone released a 720-bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with lambda GRM-B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti-(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM-B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM-B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318. The insert of the pGRM-B11 plasmid was used as a bona fide nucleic acid probe to screen mouse and human cDNA libraries prepared in the lambda gt11 or in the lambda gt10 vector. The first full-length cDNA clone (lambda GRH-Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH-B) is colinear with the mouse sequence cloned in the pGRM-B11 plasmid. The fragment of intermediate size (pGRH-CT) comprises the 3' end of the mRNA and the poly(A) tail while the short fragment (pGRH-NT) corresponds to the 5' region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.

Amino Acid Sequence↗
PubMed1990

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Transport of urate and p-aminohippurate in rabbit renal brush-border membranes.

The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.

Aminohippuric Acids↗