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M Scharf

Publications and source records attributed to M Scharf.

At least 37 records · Page 2Linked to original sources
PubMed1994

GeneQuiz: a workbench for sequence analysis.

We present the prototype of a software system, called GeneQuiz, for large-scale biological sequence analysis. The system was designed to meet the needs that arise in computational sequence analysis and our past experience with the analysis of 171 protein sequences of yeast chromosome III. We explain the cognitive challenges associated with this particular research activity and present our model of the sequence analysis process. The prototype system consists of two parts: (i) the database update and search system (driven by perl programs and rdb, a simple relational database engine also written in perl) and (ii) the visualization and browsing system (developed under C++/ET++). The principal design requirement for the first part was the complete automation of all repetitive actions: database updates, efficient sequence similarity searches and sampling of results in a uniform fashion. The user is then presented with "hit-lists" that summarize the results from heterogeneous database searches. The expert's primary task now simply becomes the further analysis of the candidate entries, where the problem is to extract adequate information about functional characteristics of the query protein rapidly. This second task is tremendously accelerated by a simple combination of the heterogeneous output into uniform relational tables and the provision of browsing mechanisms that give access to database records, sequence entries and alignment views. Indexing of molecular sequence databases provides fast retrieval of individual entries with the use of unique identifiers as well as browsing through databases using pre-existing cross-references. The presentation here covers an overview of the architecture of the system prototype and our experiences on its applicability in sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Prediction of protein structure by evaluation of sequence-structure fitness. Aligning sequences to contact profiles derived from three-dimensional structures.

The problem of protein structure prediction is formulated here as that of evaluating how well an amino acid sequence fits a hypothetical structure. The simplest and most complicated approaches, secondary structure prediction and all-atom free energy calculations, can be viewed as sequence-structure fitness problems. Here, an approach of intermediate complexity is described, which involves; (1) description of a protein structure in terms of contact interface vectors, with both intra-protein and protein-solvent contacts counted, (2) derivation of sequence preferences for 2 up to 29 contact interface types, (3) generation of numerous hypothetical model structures by placing the input sequence into a large set of known three-dimensional structures in all possible alignments, (4) evaluation of these models by summing the sequence preferences over all structural positions and (5) choice of predicted three-dimensional structure as that with the best sequence-structure fitness. Evolutionary information is incorporated by using position-dependent core weights derived from multiple sequence alignments. A number of tests of the method are performed: (1) evaluation of cyclic shifts of a sequence in its native structure; (2) alignment of a sequence in its native structure, allowing gaps; (3) alignment search with a sequence or sequence fragment in a database of structures; and (4) alignment search with a structure in a database of sequences. The main results are: (1) a native sequence can very well find its native structure among a large number of alternatives, in correct alignment; (2) substructures, such as (beta alpha)n units, can be detected in spite of very low sequence similarity; (3) remote homologous can be detected, with some dependence on the set of parameters used; (4) contact interface parameters are clearly superior to classical secondary structure parameters; (5) a simple interface description in terms of just two states, protein-protein and protein-water contacts, performs surprisingly well; (6) the use of core weights considerably improves accuracy in detection of remote homologues; (7) based on a sequence database search with a myoglobin contact profile, the C-terminal domain of a viral origin of replication binding protein is predicted to have an all-helical fold. The sequence-structure fitness concept is sufficiently general to accommodate a large variety of protein structure prediction methods, including new models of intermediate complexity currently being developed.

Amino Acid Sequence↗
PubMed1993

High yield fermentation and purification of Tendamistat disulphide analogues secreted by Streptomyces lividans.

In our studies of structure-function correlation of polypeptides we used Tendamistat (TM), an alpha-amylase-inhibitor of Streptomyces tendae, as a model to investigate the influence of different mutants on the expression and secretion of the protein. In addition, we examined the influence of replacing the two disulphide-bridges that stabilize the two-loop structure of the whole protein. The single mutants C27S, C27T, C45A, the double mutants C11A/C27A, C11A/C27S, C11A/C27T, C11A/C27L, C45/C73A and the fourfold mutant C11A/C27A/C45A/C73A were prepared. The mutated TM gene was expressed in S. lividans TK 24, which secretes the active form of the inhibitor into the culture medium. Compared with the wild-type, the double-mutated TM derivatives show an increase in secreted protein by a factor of two to ten. In contrast, the single-mutated inhibitor analogues show the reverse effect. In order to examine the influence of temperature and culture media on the production of protein derivative we used the most unstable C11A analogue. Our expression studies at 10, 19, 28 and 37 degrees C established 19 degrees C as the optimal temperature for production of the protein derivatives. The correlation between the stability and secretion of TM is discussed in the context of our knowledge of protein translocation in bacteria. Based on these experiments we optimized the fermentation parameters, isolated TM analogous on a large scale, and verified them.

Amino Acid Sequence↗
PubMed1992

Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity.

Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.

Amino Acid Sequence↗
PubMed1992

Selection of representative protein data sets.

The Protein Data Bank currently contains about 600 data sets of three-dimensional protein coordinates determined by X-ray crystallography or NMR. There is considerable redundancy in the data base, as many protein pairs are identical or very similar in sequence. However, statistical analyses of protein sequence-structure relations require nonredundant data. We have developed two algorithms to extract from the data base representative sets of protein chains with maximum coverage and minimum redundancy. The first algorithm focuses on optimizing a particular property of the selected proteins and works by successive selection of proteins from an ordered list and exclusion of all neighbors of each selected protein. The other algorithm aims at maximizing the size of the selected set and works by successive thinning out of clusters of similar proteins. Both algorithms are generally applicable to other data bases in which criteria of similarity can be defined and relate to problems in graph theory. The largest nonredundant set extracted from the current release of the Protein Data Bank has 155 protein chains. In this set, no two proteins have sequence similarity higher than a certain cutoff (30% identical residues for aligned subsequences longer than 80 residues), yet all structurally unique protein families are represented. Periodically updated lists of representative data sets are available by electronic mail from the file server "netserv@embl-heidelberg.de." The selection may be useful in statistical approaches to protein folding as well as in the analysis and documentation of the known spectrum of three-dimensional protein structures.

Algorithms↗
PubMed1992

Comprehensive sequence analysis of the 182 predicted open reading frames of yeast chromosome III.

With the completion of the first phase of the European yeast genome sequencing project, the complete DNA sequence of chromosome III of Saccharomyces cerevisiae has become available (Oliver, S. G., et al., 1992, Nature 357, 38-46). We have tested the predictive power of computer sequence analysis of the 176 probable protein products of this chromosome, after exclusion of six problem cases. When the results of database similarity searches are pooled with prior knowledge, a likely function can be assigned to 42% of the proteins, and a predicted three-dimensional structure to a third of these (14% of the total). The function of the remaining 58% remains to be determined. Of these, about one-third have one or more probable transmembrane segments. Among the most interesting proteins with predicted functions are a new member of the type X polymerase family, a transcription factor with an N-terminal DNA-binding domain related to GAL4, a "fork head" DNA-binding domain previously known only in Drosophila and in mammals, and a putative methyltransferase. Our analysis increased the number of known significant sequence similarities on chromosome III by 13, to now 67. Although the near 40% success rate of identifying unknown protein function by sequence analysis is surprisingly high, the information gap between known protein sequences and unknown function is expected to widen and become a major bottleneck of genome projects in the near future. Based on the experience gained in this test study, we suggest that the development of an automated computer workbench for protein sequence analysis must be an important item in genome projects.

Acetolactate Synthase↗
PubMed1992

Recombinant human granulocyte-macrophage colony-stimulating factor stimulates superoxide anion and hydrogen peroxide production in human neutrophils.

The effect of purified recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the oxidative metabolism of human peripheral blood granulocytes was investigated. The respiratory burst of granulocytes was assessed in individual cells by flow cytometry utilizing the oxidation of the nonfluorescent 2',7'-dichlorofluorescein (DCFH) to the highly fluorescent DCF by hydrogen peroxide (H2O2). Treatment with GM-CSF caused granulocytes to produce H2O2 without addition of a second stimulus. The amount of H2O2 produced correlated with the concentration of GM-CSF administered. Also, GM-CSF did not prime the granulocytes for enhanced H2O2 production in response to N-formylmethionyl-leucyl-phenylalanine (f-MLP). Consecutive stimulation of granulocytes with GM-CSF and f-MLP resulted in additive production of H2O2. GM-CSF also induced granulocytes to release superoxide anion (O2-) in a dose-dependent manner, when the respiratory burst was assessed by a conventional cytochrome c reduction assay. In contrast to hydrogen superoxide production, GM-CSF significantly (p < 0.001) enhanced f-MLP-stimulated release of superoxide anion over that expected from the additive effects of the two agonists.

Amino Acid Sequence↗
PubMed1990

Cortisol, ACTH, and beta-endorphin after dexamethasone administration in Parkinson's dementia.

The dexamethasone suppression test (DST) has been suggested as an effective tool for differentiating between depression and dementia. After administering 1 mg dexamethasone, we measured cortisol, ACTH, and beta-endorphin levels in 32 nondepressed patients with idiopathic Parkinson's disease (PD) (14 also with dementia) and 20 healthy, age-matched controls. Four of the 20 controls, 9 of the 18 with PD alone, and 8 of the 14 with PD and dementia were dexamethasone nonsuppressors (cortisol value greater than or equal to 5 micrograms/100 ml). PD patients without dementia (nonsuppressors) showed higher basal plasma values of cortisol (22.06 +/- 5.30 micrograms/100 ml) compared with the suppressors (13.38 +/- 3.30 micrograms/100 ml). Plasma ACTH and beta-endorphin responded in a coupled way to dexamethasone challenge. Higher basal levels of both peptides were found among PD patients (demented and nondemented), nonresponders to DST. Thus, the DST does not appear to be effective in differentiating between depression and dementia in PD. In addition, PD nonsuppressors showed higher basal values of plasma ACTH, beta-endorphin, and cortisol (similar to patients with major depression). This suggests that although the depression is clinically undetectable, both disorders may share some pathophysiological features at the hypothalamic hypophyseal adrenal level.

Adrenocorticotropic Hormone↗
PubMed1990

Bromocriptine blood levels after the concomitant administration of levodopa, amantadine and biperiden in Parkinson's disease.

We recently demonstrated that when different drugs (mainly used for the treatment of Parkinson's disease) are administered in combination they interfere with the availability of bromocriptine in the brain of rats (striatum and hypothalamus). In the present study performed with parkinsonian patients, we measured plasma levels of bromocriptine (RIA) over 4 h after giving orally 5 mg bromocriptine alone; together with levodopa 250 mg plus 25 mg DCI (10 patients); with 100 mg amantadine HCl (5 patients) and with biperiden 5 mg (5 patients). Amantadine and biperiden did not interfere with the pharmacokinetics of bromocriptine. However, levodopa significantly diminished plasma levels (a mean increment of 1.78 mg +/- 0.30 vs 0.92 +/- 0.18 mg/ml). We postulate that levodopa may interfere with the metabolism of bromocriptine in the liver. Although we did not observe substantial clinical differences among the patients (Webster scale), this study supports our previous findings and suggests that one of the advantages of combined treatment may result from a modification of the plasma levels of bromocriptine by levodopa. A "smoothing" of the plasma bromocriptine curve possibly avoids sudden oscillations of the drug availability and enables a more "stable" penetrability of the medication into the central nervous system.

Administration, Oral↗
PubMed1989

Primary structures of new 'iso-hirudins'.

Crude hirudin (12.7 U/micrograms), a complex mixture of polypeptides obtained from the leech, could be separated by microbore HPLC. A combination of amino acid analysis, N-terminal microsequencing and chemical as well as enzymatic fragmentation made the primary sequence of the new isohirudins Ia-IIIb' accessible. The biological activity determined in the thrombin inhibition test showed a comparable value for all of these compounds. The results presented address the question as to whether these isohirudins are true mutations from a family of genes or a family of leeches.

Amino Acid Sequence↗
PubMed1989

The influence of levodopa in the pharmacokinetics of bromocriptine in Parkinson's disease.

The administration of bromocriptine in addition to levodopa in Parkinson's disease produces beneficial results. Several hypotheses have explained the advantage of the combined treatment by a pharmacodynamic interaction in the striatum. However, no study has considered the possibility that levodopa modifies the kinetics of bromocriptine. In the present study performed with parkinsonian patients, we measured blood levels of bromocriptine (by radioimmunoassay) at 0, 30, 60, 90, 120, 180, and 240 min after the oral administration of bromocriptine alone and together with 250 mg levodopa plus 25 mg DCI. After loading of bromocriptine alone, we found mean peak levels at 60 min (1.42 ng/ml) and at 90 min (1.82 ng/ml). These values were reduced by levodopa (0.97 ng/ml at 60 min and 0.93 ng/ml at 90 min). Although we did not observe substantial clinical differences among the groups after the drug challenge (Webster scale), this study supports our previous findings and suggests that one of the advantages of a combined treatment may result from a modification of the plasma levels of bromocriptine by levodopa. A "smoothing" of the plasma bromocriptine curve possibly avoids sudden oscillations of the drug and enables a more "stable" penetrability of the medication into the central nervous system. Therefore long-term combined treatment is advised in preference to bromocriptine alone.

Adjuvants, Pharmaceutic↗
PubMed1989

Ultrasound assessment of the bladder volume after anterior colporrhaphy.

In 40 gynecological patients 44 different determinations of the bladder volume were made using ultrasonic methods. The product of bladder depth, height, and width, as determined from transverse and sagittal scans, showed the best correlation with the bladder volume measured by urethral catheter (r = 0.981). For 73% of the measurements the error was under 20% when the true bladder volume was greater than 100 cm3. This method gives a reasonable assessment of the bladder residual volume. It is quick, safe, and repeatable and, therefore, useful in postoperative clinical practice.

Adult↗
PubMed1988

Two-dimensional NMR studies of the porcine muscle adenylate kinase.

Porcine adenylate kinase was subjected to one- and two-dimensional proton NMR studies in order to identify amino acid spin systems and obtain sequence-specific resonance assignments. With a combination of results from a map of side-chain distances resulting from the refined X-ray crystallographic data and nuclear Overhauser effect spectroscopy (NOESY), assignments are suggested for all the aromatic spin systems.

Adenylate Kinase↗
PubMed1987

Human blood basophils display a unique phenotype including activation linked membrane structures.

To evaluate the membrane marker profile of human basophils a panel of well-established monoclonal antibodies (MoAbs, n = 60) was used for a combined toluidine/immunofluorescence staining procedure. Myeloid-associated MoAbs (particularly MoAbs against the LFA-1 family (CD11, CDw18), MoAbs directed against lactosylceramide (CDw17), anti-glycoprotein (gp) 150 MoAbs MCS 2 and MY 7 (CDw13), anti-gp 67 MoAb MY 9, anti Fc gamma-receptor (mol wt 40 kd) MoAb CIKM5, anti-CR 1 MoAb E 11, and the antiglycolipid MoAb VIM-2) were reactive with basophils, indicating a close relationship to other mature myeloid cells. Under normal conditions, basophils surprisingly express at least three activation-linked structures not detectable on mature neutrophils, ie, the p45 structure defined by MoAbs OKT-10 and VIP-2b, the p24 structure identified by the CD9 MoAb BA-2, and the receptor for interleukin 2 (IL 2) recognized by three different MoAbs (anti-TAC, IL2RI, anti-IL 2). Moreover, under short-term culture conditions basophils both in mononuclear cell (MNC) suspension and as purified fractions display the HLA-DR and T4 antigens. The neutrophilic/eosinophilic structure 3-fucosyl-N-acetyllactosamine is expressed on basophils only after neuraminidase treatment. Basophils were not stained at all by CD 16 MoAbs directed against the Fc gamma-receptor (mol wt 50 to 70 kd) of neutrophils, by the MoAb 63D3 (CDw12) recognizing the monocyte/granulocyte-associated p 200 antigen, and by the CDw 14 antibodies (VIM-13, Mo 2) defining the monocyte-specific structure p 55. Enriched basophils freshly obtained from chronic granulocytic leukemia (CGL) patients yielded identical results in FACS analyses. In summary, these data indicate that basophils generate a unique combination of surface determinants and possibly represent an activated cell population.

Antibodies, Monoclonal↗