Biomedical subjects

G Krauss

Publications and source records attributed to G Krauss.

At least 37 records · Page 2Linked to original sources

PubMed1998

Equine infectious anemia virus transactivator is a homeodomain-type protein.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.

Animals↗

PubMed1998

Effective treatment of advanced breast cancer with vinorelbine, 5-fluorouracil and l-leucovorin plus human granulocyte colony-stimulating factor.

A phase II trial was performed to investigate the efficacy and tolerance of vinorelbine (VNB), 5-fluorouracil (5-FU), l-leucovorin (LLV) and recombinant human granulocyte colony-stimulating factor (G-CSF) in advanced breast cancer. Between August 1994 and October 1996, 53 patients entered this trial. Thirty-seven patients were previously untreated and 16 patients had failed previous palliative chemotherapy with (n = 12) or without anthracyclines (n = 4). Therapy consisted of VNB 40 mg m(-2) diluted in 250 ml of saline infused over 30 min on days 1 and 14 and LLV 100 mg m(-2) administered by intravenous bolus injection and 5-FU 400 mg m(-2) diluted in 500 ml of saline infused over 2 h, both given on days 1-5 every 4 weeks. G-CSF was administered at 5 microg kg(-1) day(-1) subcutaneously on days 6-10 during each cycle. Treatment was continued in cases of response or stable disease until a total of six courses were completed. The overall response rate was 59% for chemotherapeutically naive patients (95% confidence interval 42-75%), including five complete responses (CR; 13%) and 17 partial responses (PR; 46%); ten patients (27%) had stable disease (SD) and only five (14%) progressed (PD). Second-line chemotherapy with this regimen resulted in 3/16 (19%) objective remissions, but nine patients had SD and four had PD. The median time to progression was 10.5 months (range 2-23) in previously untreated patients and 7.0 months (range 2-19) in those who had failed prior chemotherapy. After a median follow-up time of 14 months, 29 patients (55%) are still alive with metastatic disease; median survival has not been reached yet. The dose-limiting toxicity was myelosuppression: WHO grade III and IV neutropenia occurred in 15 (28%) and four patients (8%), and was complicated by septicaemia in two; grade III anaemia and thrombocytopenia were noted in four (8%) and three (6%) patients respectively. Severe (WHO grade 3) non-haematological toxicities included stomatitis in 6% and nausea/vomiting and alopecia in 2% each. Our data suggest that the combination of vinorelbine, 5-fluorouracil and l-leucovorin plus G-CSF is an effective first line regimen for treatment of advanced breast cancer. Overall toxicity was modest, with myelosuppression being the dose-limiting side-effect. Other severe adverse reactions were uncommon.

Adult↗

PubMed1998

Painful stimuli evoke potentials recorded over the human anterior cingulate gyrus.

Clinical studies of cingulotomy patients and imaging studies predict that the human cingulate gyrus might display pain-related activity. We now report potentials evoked by painful cutaneous stimulation with a CO2 laser (LEP) and recorded from subdural electrodes over the medial wall of the hemisphere. In response to facial laser stimulation on both sides, a negative (latency 211-242 ms) and then a positive wave (325-352 ms) were recorded from the cortex of right medial wall and from the falcine dura overlying the left medial wall. Medial wall LEPs were similar to scalp LEPs and were largest over the anterior cingulate and superior frontal gyri just anterior to motor cortex contralateral to the side of stimulation. These results demonstrate that there is significant direct nociceptive input to the human anterior cingulate gyrus (Brodmann's area 24).

Adult↗

PubMed1997

Imaging of delta- and mu-opioid receptors in temporal lobe epilepsy by positron emission tomography.

The involvement of opioid neurotransmitter systems in seizure mechanisms is well documented. In previous positron emission tomography (PET) studies in patients with unilateral temporal lobe epilepsy, we have found evidence for differential regulation of the opioid-receptor subtypes. The present study extends our previous observations to delta-opioid receptors by using the delta-receptor-selective antagonist [11C]methylnaltrindole ([11C]MeNTI). Paired measurements of delta- and mu-opioid receptor binding and metabolic activity were performed with PET using [11C]MeNTI and [11C]carfentanil ([11C]CFN) and [18F]fluorodeoxyglucose ([18F]FDG), respectively. Binding of [11C]MeNTI and [11C]CFN increased and [18F]FDG uptake decreased in the temporal cortex (TC) ipsilateral to the focus. Decreases in [18F]FDG uptake were more widespread regionally than were increases in opioid receptors. Increases in the delta- and mu-receptor binding showed different regional patterns. Increases in mu-receptor binding were confined to the middle aspect of the inferior TC, whereas binding of delta receptors increased in the mid-inferior TC and anterior aspect of the middle and superior TC. The increase in delta receptors suggests their anticonvulsant action, as previously shown for the delta-receptor subtype, whereas the different regional pattern of receptor alterations suggest the distinct roles of different opioid-receptor subtypes in seizure phenomena.

Adult↗

PubMed1997

Homology modeling of adenylosuccinate synthetase from Saccharomyces cerevisiae reveals a possible binding region for single-stranded ARS sequences.

Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques. The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms. The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.

Adenylosuccinate Synthase↗

PubMed1996

Enzymatic properties and inhibition by single-stranded autonomously replicating sequences of adenylosuccinate synthase from Saccharomyces cerevisiae.

Adenylosuccinate synthase (ASS) from Saccharomyces cerevisiae has been shown to bind specifically to the T-rich side of the autonomously replicating sequence (ARS) core consensus sequence [Zeidler, R., Hobert, O., Johannes, L., Faulhammer, H. & Krauss, G. (1993) J. Biol. Chem. 268, 20191-20197]. We have cloned and sequenced the gene for ASS and have studied in detail the enzymatic properties and DNA-binding activity of ASS. The deduced amino acid sequence of the yeast ASS is highly similar to the same enzymes from other sources from which it is however distinguished by its more basic nature. We show that the enzymatic activity of ASS is inhibited in a highly specific manner by the binding of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Other nucleic acids, rNTP and dNTP are not able to mimic the specific inhibitory effect. Single-base substitutions in the ARS core sequence lead to a tenfold reduction in inhibition. The inhibition data corroborate the earlier report on the DNA-binding specificity of this enzyme. The homologous enzymes from Escherichia coli and Dictyostelium discoideum do not show specific binding to single-stranded ARS sequences and their enzymatic activity is not influenced by the presence of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Treatment of ASS with alkaline phosphatase leads to a loss of DNA binding and to a loss of the inhibition by DNA of the enzymatic activity which suggests that the DNA-binding activity but not the enzymatic activity may be regulated by the phosphorylation status of the protein.

Adenylosuccinate Synthase↗

PubMed1996

Comparative studies of pyruvate kinase from PSE and normal pig muscles.

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant, Km, for phosphoenol pyruvate and a more than ten times higher Kcat/Km value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower Km value and a 100-fold increased kcat/Km value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.

Animals↗

PubMed1995

Thrombomodulin and ristocetincofactor in homocystinuria: a study in two siblings.

Homocystinuria due to cystathionine-beta-synthase deficiency (CBS-def-HOCY) initially often presents with vascular disorders, e.g. thromboembolic events. The measurement of vascular endothelial markers in plasma could help to assess endothelial damage. We determined von Willebrand factor (measured as Ristocetincofactor, RiCoF) and thrombomodulin (TM), two endothelial cell markers to our knowledge not measured systematically before in homocystinuria patients in a longitudinal study of two homocystinuric patients: Patient1 with thromboembolic disease and his asymptomatic sister, patient2. Before start of therapy in patient 1, TM and RiCoF levels both were increased. In patient 2 a moderately elevated RiCoF and a normal level of TM were found. Vitamin therapy with 15 mg folate and 600 mg pyridoxine per day led to almost complete normalization of amino acids in urine and plasma, and complete normalization of RiCoF and TM levels in both patients. Thus, TM and RiCoF elevations demonstrate that CBS-def-HOCY leads to endothelial cell damage, which resolved under vitamin therapy in the patients studied.

Adult↗

PubMed1994

Coagulation factors and markers of activation of coagulation in homocystinuria (HOCY): a study in two siblings.

Homocystinuria due to cystathionine-beta-synthase deficiency (CBS-def-HOCY) initially often present with thromboembolic events. In most cases in which coagulation factors have been analysed, a deficiency of AT-IIIc and factor VIIc has been reported, the cause of which has not been elucidated. Activation of coagulation with consumption of coagulation factors has been postulated as the mechanism. This paper reports a longitudinal study of two patients: patient 1 with thromboembolic disease and his asymptomatic sister, patient 2. Before start of therapy in patient 1, a reduction of FVIIc, other coagulation factors, and AT-IIIc was found. Markers of activation of coagulation (F1 + 2, TAT, FM, D-dimers) were elevated only in patient 1, and only at the time of thrombotic complications. In patient 2 reduced levels of FVIIc and other coagulation proteins, and a low borderline AT-IIIc level was found. Thus, in the two patients, sustained activation of coagulation can be reasonably excluded to be the cause of low levels of coagulation proteins. Vitamin therapy with 15 mg folate and 600 mg pyridoxine per day led to almost complete normalization of amino acids in urine and plasma. Thrombosis has not recurred to date. FVIIc and the other coagulation proteins and AT-IIIc increased in parallel with the biochemical remission. Direct inhibition of the activity of AT-III and coagulation factor VIII and other factors by homocysteine was attempted in vitro but could not be shown at HC concentrations known to occur in the plasma of HOCY patients. Therefore, in these patients, deficient synthesis of coagulation factors and AT-III due to a disturbance of amino acid metabolism is still the most probable explanation for the observed low levels.

Adult↗

PubMed1993

Characterization of two novel single-stranded DNA-specific autonomously replicating sequence-binding proteins from Saccharomyces cerevisiae, one of which is adenylosuccinate synthetase.

We report here the identification and characterization of two novel proteis from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highly specific manner. The two proteins, 40 and 45 kDa in size, can be distinguished by multiple criteria from each other and from the 65-kDa ssArS-T-binding protein identified recently in our laboratory (Schmidt, A. M. A., Herterich, S. U., and Krauss, G. (1991) EMBO J. 10, 981-985). The specificity of binding is inferred from gel shift and nuclease-footprinting experiments using single-stranded probes containing the core consensus ARS. With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed. Partial amino acid sequencing and enzymatic assays identify the 45-kDa protein as adenylosuccinate synthetase, an enzyme necessary for the de novo synthesis of adenylate.

Adenylosuccinate Synthase↗

PubMed1992

Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA.

The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay. A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600. These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.

Base Sequence↗

PubMed1991

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.

Base Sequence↗

PubMed1990

Resection of the epileptogenic area in critical cortex with the aid of a subdural electrode grid.

Electrode grids were implanted subdurally in 28 patients with epilepsy. In 16 of the 28 patients, an epileptogenic area was located in the speech-dominant left temporal lobe. Recordings made with the grid revealed that the epileptogenic areas in the patients varied widely in extent: the area was confined within the first 10 mm of the temporal lobe in some patients or it was scattered throughout the entire anterior to posterior 80-mm extend in others. Resection of the epileptogenic area was adjusted accordingly in each case. In 6 of 16 patients who were left-hemisphere-dominant for language, up to 55-80 mm from the tip of the temporal lobe was removed, a measure that exceeds the conventional limit of 50 mm from the tip of the dominant hemisphere. In the remaining 12 of the 28 patients, epileptogenic areas were located in a combination of several lobes. In 7 of these 12 patients, the epileptogenic area encompassed the rolandic area; it was removed without deficit in 4 patients and with expected deficit in 3. Of the latter 3 patients, 1 patient underwent hemispherectomy, and a large portion of the epileptogenic rolandic cortex in the frontal and parietal lobes was removed from the other 2. There were 2 cases of grid-related infection, which cleared with antibiotic treatment; there were no lasting complications of grid implantation in any patient. There was no mortality. Electroencephalographic recording and functional mapping using subdural electrode grids allow a tailored, maximal resection of epileptogenic tissue with minimal injury to critical cortex.

Brain Diseases↗

PubMed1989

The fidelity of DNA polymerases during in vitro replication of a template containing 5-bromouracil at a specific site.

The miscoding properties of a 5-bromodeoxyuridine (dB) containing DNA template during in vitro replication have been investigated. 5-bromodeoxyuridine was introduced site-specifically into the amber 16 codon of a 25-mer oligodeoxynucleotide representing part of the sequence of phi x174am16(+)DNA. The dB containing oligodeoxynucleotide served as a template for in vitro replication by DNA polymerase alpha, DNA polymerase I (Escherichia coli) and AMV reverse transcriptase. The amber 16 revertant assay was used to detect the presence of misincorporated bases in the replication products. For all three DNA polymerases, the presence of dB does not constitute a significant barrier to replication. Errors at the position of dB substitution were found to originate exclusively from dGTP:dB mispairing during in vitro replication thus inducing A-T----G-C transitions. The dGTP:dB mismatches are formed at a 2-4-fold higher frequency as compared to dGTP:T mismatches. Our results indicate that the miscoding potential of dB-substituted DNA templates during replication is only weak at the specific site observed.

Avian Myeloblastosis Virus↗

PubMed1988

An evaluation of the SCM test for the diagnosis of cancer of the breast.

The SCM test (structuredness of the cytoplasmic matrix) consists of measuring the fluorescence polarization of fluorescein which is introduced into a particular sub-group of peripheral lymphocytes. The test has a non-specific part for the general detection of cancer and a specificity procedure which is based on the use of specific cancer extracts. In this article we deal only with the latter in relation to breast cancer. Blood samples from 94 patients have been tested; six of these had mastectomy performed previously; 83 underwent consecutively a surgical procedure and histology was obtained; five were only clinically examined. In 45/49 (92%) patients, correlation between a positive specificity test and tissue malignancy was found. Out of 35 patients with non-malignant proliferative lesions (as found by histology), 25 reacted positively in the test. Two out of five patients with non-malignant, non-proliferative lesions reacted positively in the test. Five patients who were defined as normals by clinical examination reacted negatively in the test. These results indicate the potential of the SCM test for detecting breast malignancy. The clinical implications of the test for cancer diagnosis are discussed.

Adolescent↗

PubMed1988

Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene.

Porin F is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. It forms water-filled pores of variable size. Porin F is a candidate for a vaccine against P. aeruginosa because it antigenically cross-reacts in all serotype strains of the International Antigenic Typing Scheme. We have isolated the gene for porin F from a lambda EMBL3 bacteriophage library by using oligodeoxynucleotide hybridization probes and have determined its nucleotide sequence. Different peptide sequences obtained from isolated porin F confirmed the deduced protein sequence. The mature protein consists of 326 amino acid residues and has a molecular weight of 35,250. The precursor contains an N-terminal signal peptide of 24 amino acid residues. S1 protection and primer extension experiments, together with Northern (RNA) blots, indicate that the mRNA coding for porin F is monocistronic with short untranslated regions of about 58 bases at the 5' end and about 47 bases at the 3' end. The sequences in the -10 and -35 regions upstream of the transcriptional start site are closely related to the Escherichia coli promoter consensus sequences, which explains why the porin F gene is expressed in E. coli under the control of its own promoter. The amino acid sequence of porin F is not homologous to the different E. coli porins OmpF, OmpC, LamB, and PhoE. On the other hand, a highly homologous region of 30 amino acids between the OmpA proteins of different enteric bacteria and porin F of P. aeruginosa was detected. The core region of the homology to E. coli OmpA had 11 of 12 amino acid residues in common.

Amino Acid Sequence↗

PubMed1987

The cleavage of single-stranded DNA by the isoschizomeric restriction endonuclease HhaI and CfoI.

The cleavage of single-stranded (ss) M13mp8(+) DNA by the isoschizomeric restriction endonucleases HhaI and CfoI has been investigated. The two enzymes differ considerably in their ability to cleave ssDNA. HhaI cleaves ssDNA about two orders of magnitude faster than does CfoI, although both enzymes show the same activity when assayed on double-stranded DNA. From the cleavage of oligonucleotides and of M13mp8(+) DNA fragments it is concluded that cleavage of ssDNA occurs via transiently formed double-stranded hairpin structures. A rough correlation exists between the stability of the secondary structures and the cleavage efficiency.

Coliphages↗

PubMed1986

Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA.

The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA. Errors leading to a loss of alpha-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86% of the errors whereas base deletions amount to 12%. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.

Animals↗