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M Härtlein

Publications and source records attributed to M Härtlein.

At least 19 recordsLinked to original sources
PubMed2002

Gemcitabine monotherapy as second-line treatment in cisplatin-refractory transitional cell carcinoma - prognostic factors for response and improvement of quality of life.

OBJECTIVES: i) To evaluate objective response, toxicity, and quality of life (QoL) of gemcitabine monotherapy as second-line treatment in patients with cisplatin-refractory, metastatic transitional cell carcinoma (TCC). ii) To assess prognostic parameters for response to treatment and for improvement of QoL parameters. PATIENTS AND METHODS: 30 patients were prospectively enrolled in this open-label, nonrandomized multicenter phase II trial. Patients received up to 6 courses of gemcitabine monotherapy (1,250 mg/m(2) on day 1 and 8 of a 21-day course). 28 of 30 patients were available for response evaluation. RESULTS: Objective response (OR) was seen in 3/28 (11%) of patients (2 complete remissions, 1 partial remission). The mean time to progression (TTP) was 4.9 +/- 3.5 months and mean disease-specific survival time was 8.7 +/- 4.7 months. 13 of 28 patients did not progress (OR + 10 stable diseases), and TTP (8.0 +/- 2.7 months, p < 0.001) as well as survival time (10.2 +/- 3.8 months, p < 0.05) differed significantly from those who showed progressive disease within 18 weeks of treatment. Pain values significantly improved in the group of responders from 4.3 +/- 1.9 to 5.8 +/- 1.3 points (p < 0.05). Response to cisplatin pretreatment was the best prognosticator for the response to gemcitabine. CONCLUSIONS: Gemcitabine monotherapy as second-line treatment is justified in patients with metastatic TCC who are refractory to cisplatin treatment. Patients with initially OR to cisplatin benefit most from second-line treatment. QoL remains stable during treatment, and pain improves especially in patients with bone metastases.

Carcinoma, Transitional Cell↗
PubMed2001

Human anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS) increase the affinity of the enzyme for its tRNA substrate.

Autoantibodies directed against specific human aminoacyl-tRNA synthetases have been associated with a clinical picture including myositis, arthritis, interstitial lung disease and other features that has been referred to as the "anti-synthetase syndrome". Anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS), the most recently described anti-synthetase autoantibodies, are directed against human cytosolic asparaginyl-tRNA synthetase and neutralize specifically its activity. Here we show that these antibodies recognize two epitopes on the human enzyme, an N-terminal epitope reactive in immunoblot experiments and a heat-labile epitope in the catalytic domain. In contrast to the well studied anti-Jo-1 autoantibodies anti-KS when bound to the synthetase increase the affinity of the synthetase for its tRNA substrate and prevent aminoacylation without interfering with the amino acid activation step.

Acylation↗
PubMed1998

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Adenosine Triphosphate↗
PubMed1998

Analysis and overexpression in Escherichia coli of a staphylococcal gene encoding seryl-tRNA synthetase.

We have sequenced and expressed in Escherichia coli the gene encoding the seryl-tRNA synthetase from the pathogenic bacterium Staphylococcus aureus. The overexpressed and purified recombinant enzyme was able to aminoacylate unfractionated tRNA from E. coli. Its activity was not affected by antibodies raised against and inhibiting the E. coli seryl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1998

Human cytosolic asparaginyl-tRNA synthetase: cDNA sequence, functional expression in Escherichia coli and characterization as human autoantigen.

The cDNA for human cytosolic asparaginyl-tRNA synthetase (hsAsnRSc) has been cloned and sequenced. The 1874 bp cDNA contains an open reading frame encoding 548 amino acids with a predicted M r of 62 938. The protein sequence has 58 and 53% identity with the homologous enzymes from Brugia malayi and Saccharomyces cerevisiae respectively. The human enzyme was expressed in Escherichia coli as a fusion protein with an N-terminal 4 kDa calmodulin-binding peptide. A bacterial extract containing the fusion protein catalyzed the aminoacylation reaction of S.cerevisiae tRNA with [14C]asparagine at a 20-fold efficiency level above the control value confirming that this cDNA encodes a human AsnRS. The affinity chromatography purified fusion protein efficiently aminoacylated unfractionated calf liver and yeast tRNA but not E.coli tRNA, suggesting that the recombinant protein is the cytosolic AsnRS. Several human anti-synthetase sera were tested for their ability to neutralize hsAsnRSc activity. A human autoimmune serum (anti-KS) neutralized hsAsnRSc activity and this reaction was confirmed by western blot analysis. The human asparaginyl-tRNA synthetase appears to be like the alanyl- and histidyl-tRNA synthetases another example of a human Class II aminoacyl-tRNA synthetase involved in autoimmune reactions.

Acylation↗
PubMed1997

Genomic organization, cDNA sequence, bacterial expression, and purification of human seryl-tRNA synthase.

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli. Two human brain cDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5' region of the cDNA was missing. This 5' region was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25%), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA.

Amino Acid Sequence↗
PubMed1997

Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.

The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

Amino Acid Sequence↗
PubMed1997

Mitochondrial asparaginyl-tRNA synthetase is encoded by the yeast nuclear gene YCR24c.

One of the open reading frames located on yeast Saccharomyces cerevisiae chromosome III, YCR24c, appeared to code for a protein of unknown function, but the predicted sequence showed similarity with asparaginyl-tRNA synthetase from Escherichia coli, with 38% amino acid identity. There is a putative mitochondrial targeting signal at the N-terminus of the YCR24c product. Northern blot analysis of total RNA from a wild-type strain sigma1278b confirmed that YCR24c was transcribed. Disruption of the chromosomal copy of YCR24c in a respiratory-competent haploid cell induced a petite phenotype, but did not affect cell viability. This respiratory-defective phenotype is typical for a mutation in a nuclear gene that induces a non-functional mitochondrial protein synthesis system. The protein encoded by YCR24c was expressed in Escherichia coli in a histidine-tagged form and isolated. The enzyme aminoacylated unfractionated Escherichia coli tRNA with asparagine. These results identified YCR24c as the structural gene for yeast mitochondrial asparaginyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1997

Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity.

A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.

Cloning, Molecular↗
PubMed1996

Asparaginyl-tRNA synthetase from Thermus thermophilus HB8. Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli.

The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced. Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da). Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94%. The amino acid sequence of asparaginyl-tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginyl-tRNA synthetase sequences (30-55% identity). By expression of the T. thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps. The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C. Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques. The crystals diffract X-rays beyond 2.8 A.

Amino Acid Sequence↗
PubMed1995

An immunodominant antigen of Brugia malayi is an asparaginyl-tRNA synthetase.

Lymphatic filariasis is caused by infection with the filarial nematodes Brugia malayi, Brugia timori, Wuchereria bancrofti and Onchocerca volvulus which collectively infect about 200 million persons throughout the world. Protein sequence homology analysis of a major nematode antigen suggested that it was a class II aminoacyl-tRNA synthetase. The overproduction, purification and verification that the major B. malayi antigen is an asparaginyl-tRNA synthetase is described.

Amino Acid Sequence↗
PubMed1995

Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation.

Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution.

Acylation↗
PubMed1995

Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code.

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA(ser) in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 aminoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3' end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence.

Acylation↗
PubMed1995

Isolation and characterization of an Escherichia coli seryl-tRNA synthetase mutant with a large increase in Km for serine.

A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in Km for serine of seryl-tRNA synthetase is described. The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2. The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site. This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid.

Base Sequence↗
PubMed1994

Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.

Acylation↗
PubMed1993

In vivo overexpression and purification of Escherichia coli tRNA(ser).

DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.

Base Sequence↗
PubMed1992

Asparaginyl-tRNA synthetase from the Escherichia coli temperature-sensitive strain HO202. A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP.

The Escherichia coli K12 mutant gene, asnS40, coding for asparaginyl-tRNA synthetase (AsnRS) in the temperature-sensitive strain HO202, was isolated from genomic DNA using the Polymerase Chain Reaction. DNA sequencing revealed that the mutant enzyme differs from the wild-type AsnRS by two amino acids, but only the P231L replacement leads to a change in aminoacylation activity. In the ATP-PPi exchange reaction at 37 degrees C the purified P231L enzyme has a more than 50-fold increased Km value for asparagine compared to the wild-type enzyme, while the Km value for ATP is increased 8-fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased Km values for asparagine and ATP. Interestingly Pro-231 is part of the conserved motif 2 in class II aminoacyl-tRNA synthetases (Eriani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. (1990) Nature 347, 203-206), indicating that this motif might be involved in all class II enzymes in amino acid activation.

Acylation↗