PubMed Health⌕ Search

Biomedical subjects

W Schäfer

Publications and source records attributed to W Schäfer.

At least 19 recordsLinked to original sources
PubMed1992

The amino acid sequence of a type I copper protein with an unusual serine- and hydroxyproline-rich C-terminal domain isolated from cucumber peelings.

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30-37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.

Amino Acid Sequence↗
PubMed1992

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.

Amino Acid Sequence↗
PubMed1992

Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei.

A new inositol derivative could be isolated from the Archaeum Pyrococcus woesei and identified as di-myo-inositol-1,1'-phosphate by 1H, 31P NMR spectroscopy, mass spectrometry and thin layer chromatography. In P. woesei, this inositol phosphate represents the dominant counterion of K+ which ranges from 500 to 600 mM. The role of the potassium salt of di-myo-inositol-1,1'-phosphate as thermostabilizer is discussed.

Archaea↗
PubMed1992

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1992

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.

Amino Acid Sequence↗
PubMed1992

The complete primary structure of the boar spermadhesin AQN-1, a carbohydrate-binding protein involved in fertilization.

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucida-binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as well as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism.

Amino Acid Sequence↗
PubMed1992

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa.

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.

Amino Acid Sequence↗
PubMed1992

Isolation and biochemical characterization of two isoforms of a boar sperm zona pellucida-binding protein.

Protein-carbohydrate complementarity has been recognized as a general mechanism of gamete recognition and adhesion in the process of fertilization throughout the whole animal kingdom. It appears that carbohydrate-binding molecules on the anterior sperm head surface mediate the binding of the male gamete to certain glycoconjugates present on the egg's extracellular coat. Subtle differences in protein and carbohydrate conformation may confer to this interaction a species-specific character. The mechanism responsible for gamete recognition is, however, poorly understood. A step in its elucidation is the characterization of the complementary molecules on the egg and sperm surfaces. With this aim we report here the isolation and partial structural characterization of two isoforms of a zona pellucida-binding protein (which we call AWN-1 and AWN-2) from boar spermatozoa, including partial sequence determination, assignment of disulphide bonds and identification of an N-terminal blocking group. AWN-1 and AWN-2 were isolated from acid extracts of washed ejaculated sperm and were present in seminal vesicle secretions, but absent in samples of epididymal fluid, suggesting a seminal vesicle origin for these sperm proteins. No analogous protein sequence could be found in the MIPS data bank, indicating that the AWN proteins may belong to a novel mammalian protein family involved in fertilization.

Amino Acid Sequence↗
PubMed1992

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor.

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm-associated and the seminal plasma inhibitors may confer to them different physico-chemical properties which are postulated to be of functional importance.

Acrosin↗
PubMed1992

The free radical in pyruvate formate-lyase is located on glycine-734.

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.

Acetyltransferases↗
PubMed1992

Steroid receptors in ovarian carcinoma: immunohistochemical determination may lead to new aspects.

87 nonpretreated stage III/IV ovarian common epithelial carcinomas were studied for estrogen receptor (ER) and progesterone receptor (PR) content by both immunohistochemistry (IHC) and biochemical (DCC) analysis. While the DCC assay showed tumors to be receptor-positive in 62% (ER) and 66% (PR), receptor-positive malignant epithelial cells were only detected in 38% (ER) and 31% (PR) by IHC. There was only a low correlation between the semiquantitative results of ER and PR IHC and the corresponding values of DCC receptor determination. The finding of steroid receptor-positive stromal cells without any evidence of hormone receptor-positive epithelial tumor cells offers a possible explanation for discrepant results in numerous cases with obviously "false positive" results of DCC analysis. Since the considerable heterogeneity of steroid receptor expression present in many ovarian neoplasms can only be detected by IHC, it seems to be the appropriate method of ER and PR determination. Most patients were treated by both radical cytoreductive surgery (n = 76) and a platinum-based chemotherapy (n = 79). ER was not shown to be of significant prognostic value. However, survival was significantly better in patients with PR positive tumors (IHC and DCC) on univariate analysis. Residual tumor after primary surgery was the only remaining significant prognostic factor after multivariate analysis. Further studies are needed to clarify the biological function of steroid receptor-positive stromal cells in ovarian carcinomas.

Adult↗
PubMed1992

Cutinase is not required for fungal pathogenicity on pea.

Cutinase, a fungal extracellular esterase, has been proposed to be crucial in the early events of plant infection by many pathogenic fungi. To test the long-standing hypothesis that cutinase of Nectria haematococca (Fusarium solani f sp pisi) is essential to pathogenicity, we constructed cutinase-deficient mutants by transformation-mediated gene disruption of the single cutinase gene of a highly virulent N. haematococca strain. Four independent mutants were obtained lacking a functional cutinase gene, as confirmed by gel blot analyses and enzyme assays. Bioassays of the cutinase-deficient strains showed no difference in pathogenicity and virulence on pea compared to the wild type and a control transformant. We conclude that the cutinase of N. haematococca is not essential for the infection of pea.

Base Sequence↗
PubMed1992

The role of eicosanoids in reproduction.

The central role of eicosanoids in reproduction was studied in areas of important clinical interest. First, their involvement in pregnancy-induced hypertension was investigated. Urine of normotensive and hypertensive pregnant women was analysed for 6-keto-PGF1 alpha, TXB2 and PGE2 by HPLC/RIA. PGE2 and 6-keto-PGF1 alpha excretion was markedly reduced in the preeclamptic subgroup of hypertensive patients during the last two trimesters. A reduced urinary excretion of 6-keto-PGF1 alpha, TXB2 and PGE2 was also found in a hypertension animal model (rat). Further, tissue cultures of human placentas, deciduas and fetal membranes from hypertensive pregnancies displayed a reduced prostaglandin production. Secondly, in the same in-vitro model the central role of PGE2 of fetal membrane origin for the beginning or parturition was shown. Thirdly, concerning endometrial function, the enhancement of PGF2 alpha and PGE2 formation in secretory endometrial cells by estradiol-17 beta and progesterone was documented. Fourthly, lipoxygenase product content in peritoneal fluid of endometriotic patients did not differ from controls.

Animals↗
PubMed1992

[Perioperative risk of surgical therapy of Crohn disease].

301 patients, operated for Crohn's disease in a ten-year period (1980-1989) were analyzed retrospectively, regarding perioperative risk of bowel resection (n = 336). The overall mortality rate was 0.6% (elective 0, emergency 6%). The reasons of this low mortality rate are of different nature. Very important is a meticulous perioperative treatment and a standardized operative approach.

Adolescent↗
PubMed1991

The amino acid sequence of AQN-3, a carbohydrate-binding protein isolated from boar sperm. Location of disulphide bridges.

Gamete recognition and adhesion are essential steps in fertilization. Among others, carbohydrate-binding proteins on the sperm surface have been recognized to play a central role in the initial interaction of the male gamete with components of the zona pellucida of the homologous investing oocyte. We have isolated several members of a carbohydrate- and zona pellucida-binding protein family from ejaculated sperm. Here we report the biological origin and structural characterization of AQN-3, a component of this carbohydrate-binding family. The molecular weight of purified AQN-3 was determined by plasma desorption mass spectrometry. The protein was chemically and enzymatically degraded, the proteolytic fragments isolated and characterized by N-terminal sequencing and fast atom bombardment mass spectrometry. In this manner we established the complete amino acid sequence of AQN-3 and the location of its two disulphide bonds. No analogous protein sequence could be found in the MIPS protein sequence data bank, indicating that AQN-3 may belong to a novel mammalian carbohydrate-binding protein family.

Amino Acid Sequence↗
PubMed1991

Identification of the disulfide bond pattern in albolabrin, an RGD-containing peptide from the venom of Trimeresurus albolabris: significance for the expression of platelet aggregation inhibitory activity.

Albolabrin is a 73 amino acid peptide isolated from the venom of Trimeresurus albolabris. It contains an RGD sequence and 12 cysteines and is a potent inhibitor of both platelet aggregation and fibrinogen binding to the GPIIb/IIIa complex. This protein shows a high degree of analogy (primarily due to the alignment of all cysteines and the RGD) with a number of other viper venom proteins which inhibit cell adhesion and platelet aggregation and are referred to as disintegrins: rhodostomin, trigramin, flavoridin, applagin, elegantin, and batroxostatin. In this study, we found that the reduction and vinylpyridylethylation of albolabrin and flavoridin decreased their platelet aggregation inhibitory activity approximately 40-50 times. It can be postulated that the higher potency of native and reduced flavoridin as compared to albolabrin depends on the substitution of the Asp of albolabrin with a Phe at the C-terminal end of the RGD in flavoridin. The activity of a synthetic C-terminal peptide derived from flavoridin (residues 35-65) containing four cysteines was about 75 times lower than that of the original flavoridin. The substitution of a pair of cysteine residues with alanines in this peptide resulted in further loss of activity. In order to identify the disulfide bonds in albolabrin, the molecule was digested consecutively by trypsin and porcine pancreatic elastase. Peptides resulting from this digestion were isolated by reverse-phase HPLC and identified by amino acid composition and mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1991

[A prospective study for evaluating postoperative risk and long-term prognosis of mesenteric infarct].

In a prospective study on the patient material of the Surgical Clinic of the Johannes-Gutenberg-University Mainz the prognostically relevant factors in acute mesenteric vascular occlusion were investigated. From September 1, 1985 until August, 31, 1989 46 patients (24 men, 22 women) with an average age of 67.2 years were treated for an acute mesenteric vascular occlusion. Postoperatively 56.5% of the patients died. The postoperative lethality was correlated statistically significant to the period of anamnesis, the aetiology of the occlusion, the patient's age, the preoperative ASA score, and the length of the ischemically damaged bowel. The postoperative blood lactat level correlated with the outcome. Only two patients survived a second operation with a further bowel resection. One of these patients had a normal blood lactat level. The two year survival rate of the postoperative surviving patient's totalled at 70%. These results confirm the known prognostically relevant factors as well as the value of the blood lactat level in the postoperative period. They show as well, that an early second look operation depending on the intraoperative findings of the primarily operation and prior to an increase of the blood lactat level and prior to a deterioration in the patients condition is justified.

Aged↗