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B Krebs

Publications and source records attributed to B Krebs.

At least 19 recordsLinked to original sources
PubMed1998

The membrane proximal cytokine receptor domain of the human interleukin-6 receptor is sufficient for ligand binding but not for gp130 association.

Interleukin-6 (IL-6) belongs to the family of the "four-helix bundle" cytokines. The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains. Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif. On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6.IL-6R associates with the signal transducing protein gp130. The IL-6R consists of three extracellular domains. The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation. Here we have investigated the properties and functional role of the third membrane proximal domain. The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding. When complexed with IL-6, however, it fails to associate with the gp130 protein. Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling.

Antigens, CD↗
PubMed1998

Recombinant human single chain Fv antibodies recognizing human interleukin-6. Specific targeting of cytokine-secreting cells.

A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6). Two antibody-bearing phages were selected that bound IL-6. The complementary-determining region 3 loops of the variable heavy chains of these two antibodies differed in length and sequence and recognized two distinct epitopes. One of the single chain Fv fragments isolated (H1) was found to bind human (but not murine) IL-6 with an affinity comparable to that of the human IL-6 receptor. H1 also recognized newly synthesized human IL-6 intracellularly, as shown by indirect immunofluorescence. H1 did not neutralize human IL-6, and the H1 epitope was mapped to a region of IL-6 not involved in interactions with IL-6, IL-6 receptor, or the signal-transducing protein gp130. To target IL-6-secreting cells, we then constructed a bispecific antibody fragment (a diabody) comprising H1 and the antigen binding site of the T-cell activating monoclonal antibody OKT3. The diabody led to T-cell-mediated killing of cells secreting IL-6.

Antibodies, Bispecific↗
PubMed1998

A new polyoxometalate complex inhibits retrovirus encoded reverse transcriptase activity in vitro and in vivo.

We examined the recently synthesized and characterized polyoxometalate compound (NH4)10[Co2Sb2W20 O70(H2O)6] (POM1). The inhibitory potency of POM1 was studied in tissue culture experiments with uninfected and Rous sarcoma virus (RSV)-infected chicken embryo fibroblasts (CEF) in vivo. We measured a considerable decrease in total cellular phosphotyrosine content in treated infected cells in vivo. POM1 treatment of SR-RSV-A infected CEF in vivo resulted in decreased pp60v-src activity, possibly due to a reduced rate of v-src translation caused by the inhibitory effect of POM1 on the RSV encoded reverse transcriptase activity (RT) activity. In further studies we were able to demonstrate the inhibitory effect of this complex on the RT activity of RSV in vitro and in vivo.

Animals↗
PubMed1996

Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures.

Purple acid phosphatase is a widely distributed non-specific phosphomonoesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 angstroms resolution, respectively. Furthermore the resolution of the unligated enzyme, recently solved at 2.9 angstroms could be extended to 2.65 angstroms with completely new data. The binding of both oxoanions is not accompanied by larger conformational changes in the enzyme structure. Small movements with a maximal coordinate shift of 1 angstroms are only observed for the active site residues His295 and His296. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also proposed for the unligated structure a bridging hydroxide ion completes the coordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group.

Acid Phosphatase↗
PubMed1996

Different alleles of the fcrA/mrp gene of Streptococcus pyogenes encode M-related proteins exhibiting an identical immunoglobulin-binding pattern.

The majority of group A streptococci (GAS, Streptococcus pyogenes) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong either to the emm or the emm-related (fcrA/mrp and enn) gene family and are located in close proximity on the GAS genome, where they form part of the vir regulon. In the present study analysis of sequence data of the 5' terminal portions of the fcrA/mrp genes from GAS isolates representing 37 different M serotypes led to a classification of six different types. Thus, although fcrA/mrp genes exhibit an allelic polymorphism, they do not display the high degree of N-terminal sequence diversity found among emm genes. The nucleotide sequences of the fcrA/mrp genes from 3 GAS isolates, belonging to serotypes M8, M9, and M13 and representing newly characterized fcrA/mrp gene types, are reported. Analysis of the Ig-binding properties of recombinant FcrA/Mrp8, 9, and 13 proteins, demonstrated a similar Ig-binding profile being reactive with human IgG subclasses 1, 2, and 4. This pattern is identical to that previously described for other recombinant fcrA/mrp4, 49, 64/14 and 76 gene products, indicating that this property is not affected by the N-terminal variability. Evidence for recombination between an fcrA/mrp and an mga gene was observed in an M-type 33 strain isolate providing further support for the concept of gene rearrangement contributing to the diversity of vir regulon gene products.

Amino Acid Sequence↗
PubMed1995

Structural relationship between the mammalian Fe(III)-Fe(II) and the Fe(III)-Zn(II) plant purple acid phosphatases.

The primary structure of uteroferrin (Uf), a 35 kDa monomeric mammalian purple acid phosphatase (PAP) containing a Fe(III)-Fe(II) center, has been compared with the sequence of the homodimeric 111 kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (KBPAP). The alignment suggests that the amino acid residues ligating the dimetal center are identical in Uf and KBPAP, although the geometry of the coordination sphere might slightly differ. Secondary structure predictions indicate that Uf contains two beta alpha beta alpha beta motifs thus resembling the folding topology of the plant enzyme. Guided by the recently determined X-ray structure of KBPAP a tentative model for the mammalian PAP can be constructed.

Acid Phosphatase↗
PubMed1995

Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site.

Kidney bean purple acid phosphatase (KBPAP) is an Fe(III)-Zn(II) metalloenzyme resembling the mammalian Fe(III)-Fe(II) purple acid phosphatases. The structure of the homodimeric 111-kilodalton KBPAP was determined at a resolution of 2.9 angstroms. The enzyme contains two domains in each subunit. The active site is located in the carboxyl-terminal domain at the carboxy end of two sandwiched beta alpha beta alpha beta motifs. The two metal ions are 3.1 angstroms apart and bridged monodentately by Asp164. The iron is further coordinated by Tyr167, His325, and Asp135, and the zinc by His286, His323, and Asn201. The active-site structure is consistent with previous proposals regarding the mechanism of phosphate ester hydrolysis involving nucleophilic attack on the phosphate group by an Fe(III)-coordinated hydroxide ion.

Acid Phosphatase↗
PubMed1995

Acetabular penetration of sliding screw.

Case report of disengagement of sliding screw from the plate and penetration through the acetabulum into the pelvic cavity. The mechanism was probably technical failure in combination with osteoporosis and a new trauma in the region. We find that it is possible to remove the screw through the old incision, but the patient should be prepared for laparotomy.

Acetabulum↗
PubMed1994

The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing.

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(III)-Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)-Fe(II) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-C-generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.

Acid Phosphatase↗
PubMed1994

Genetic variability of the emm-related gene of the large vir regulon of group A streptococci: potential intra- and intergenomic recombination events.

One of the most prevalent genetic lineages of group A streptococci (GAS) harbors a genomic locus termed the large vir regulon, which contains an emm gene encoding the antiphagocytic M protein, and structurally related fcrA and enn (emm-related) genes encoding immunoglobulin-binding proteins. In the present study more than 100 large vir regulons from 42 different GAS serotypes were analyzed by PCR and partial DNA sequencing. On comparing these data to published sequences, sites of mutational and putative recombinational events were identified and ordered with respect to their intra/intergenic or intra/intergenomic nature. The emm-related genes were found to display small intragenic deletions or insertions, were completely deleted from, or newly inserted into the genome, or were fused to adjacent genes. Intergenomic exchanges of complete emm-related genes, or segments thereof, between different vir regulons were detected. Most of these processes seem to involve short flanking direct repeats. Occasionally, the structural changes could be correlated with changes in the functions of the encoded proteins.

Amino Acid Sequence↗
PubMed1994

The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean. Determination of the structure by a combination of matrix-assisted laser desorption/ionization mass spectrometry and selective enzymic degradation.

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110-kDa glycoprotein related to the mammalian purple acid phosphatases with a two-metal cluster at the active site contains five oligosaccharide side chains/monomer. The N-linked glycan structures were characterized by selective enzymic degradation in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The purified protein was cleaved by cyanogen bromide. One 30-kDa large methionine-free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an alpha 1-3 fucose attached to the asparagine-linked N-acetylglucosamine prevented removal of the glycan by peptide N-glycosidase F; peptide N-glycosidase A removed all carbohydrates from the peptides. To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex-type xylose-containing structure with four of the five having an additional fucose.

Acid Phosphatase↗
PubMed1994

[Isochromanylpencillins].

(R/S)-Isochroman-1-carboxylic acid (1) is resolved to its enantiomers and as well as these reacted with 6-aminopenicillanic acid to the title compounds 2. The activity of the new penicillin-derivatives against gram positive and gram negative bacteria is compared with that of benzyl penicillin.

Bacteria↗
PubMed1993

Circadian variations in the number of actively secreting sebaceous follicles and androgen circadian rhythms.

Sebum excretion has been shown to demonstrate a circadian rhythm using a gravimetric method (cigarette paper). With the newly introduced method of Sebutape, we confirmed this periodicity and showed that the elevation in sebum excretion is correlated with an increase in the number of secreting follicles. We found, furthermore, that the number of secreting follicles on the forehead showed a distinct and statistically significant circadian rhythmicity, in contrast to those of the chest, which remained almost constant. The quantification in plasma levels of cortisol, melatonin, delta-4-androstenedione, dehydroepiandrosterone sulphate, and free testosterone showed no correlation with sebum excretion at either site. These observations suggest that local factors are involved in control of sebum secretion.

Adult↗
PubMed1992

Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean.

Purple acid phosphatase from red kidney bean has been crystallized from ammonium sulfate solutions in the pH range from 3.5 to 5.5. The crystal form is tetragonal bipyramidal and the largest crystals grew up to 2.0 mm long. Systematic absences indicate one of the enantiomorphic space groups P4(1)2(1)2 (92) or P4(3)2(1)2 (96) with cell dimensions a = b = 104.1(1) A and c = 308.7(2) A. The asymmetric unit contains one dimer with Mr of 110,700, determined by ultraviolet-laser desorption mass spectrometry. The crystals, with a salt-free density of 1.12 g/cm3 and a water content of 67%, diffract to 3.5 A.

Acid Phosphatase↗
PubMed1991

Protein-chemical standardization of the erythrocyte glutathione reductase activation test (EGRAC test). Application to hypothyroidism.

The erythrocyte glutathione reductase activation coefficient (EGRAC) is an index of riboflavin deficiency or, more exactly, of FAD deficiency in man. In this report a sensitive version of the EGRAC test is introduced that is based on the molecular properties of glutathione reductase and its FAD-free apoenzyme. The hemoglobin concentration of the blood sample can be estimated simultaneously using the spectrophotometric absorption at 340 nm. - The method was tested for 33 thyroidectomized patients in comparison with a euthyroid control group. From the average EGRAC values (1.40 vs. 1.22) it was deduced that the average free FAD level was approximately 2 times lower for the patients' than for the control group. Discussed is the role of the EGRAC test in hormonal and nutritional disorders.

Adult↗
PubMed1990

[Pain and dysesthesias in the mastectomy scar].

With the object of investigating the occurrence of pain and dysaesthesiae in the scar following mastectomy, 120 were interviewed by a standard questionnaire in a prospective study. These women had commenced postoperative control or treatment in the Department of Oncology in the University Hospital of Aarhus consecutively during a one-year period. One hundred and ten of these women were interviewed again one year later. In 92% of the patients with scar pain and dysaesthesiae, these appeared within the first three months postoperatively. At the first interview, 42 patients (35%) had scar pain and 15 of these (13%) experienced constant scar pain. One year later, 25 patients (23%) still had scar pain and 13 patients (12%) had experienced persistent scar pain throughout the entire year. Two of the patients (8%) with scar pain had constant pain while, at the second interview, 14 patients (56%) reported that the scar pain lasted for seconds. Twenty-seven patients (23%) had dysaesthesiae at the first interview while 29 patients experienced these continually one year after operation. Both the intensity and the duration of the scar pain diminished with the elapse of time. This held also true where dysaesthesiae were concerned but was not so marked.

Adult↗
PubMed1990

Activation signals in human lymphocytes: interleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C.

The mitogenic activation of resting T lymphocytes involves two distinct cellular events, the synthesis of the ultimate mitogen interleukin 2 and the synthesis and expression of receptors for it. In order to get more detailed information on the mechanisms associated with these activating steps (the effects of different stimuli, leading to activation of protein kinase C were investigated in human lymphocytes). The anti-T-cell receptor (TCR) and anti-CD3 monoclonal antibodies (BMA 031 and BMA 030, respectively), as well as the combination of the phorbol ester, TPA, with a calcium ionophore-induced interleukin 2 synthesis and subsequent proliferation in human peripheral blood lymphocytes. Incubation of cells with synthetic diacylglycerols and calcium ionophores proved to be effective in expression of high affinity interleukin receptors, no detectable amounts of interleukin 2 were, however, synthetized. When diacylglycerols were, however, added repetitively, interleukin 2 was also produced. Both anti-TCR/CD3 antibodies and TPA or DiC8 caused activation and translocation of protein kinase C from the cytosol to the plasma membrane. Significant differences, however, were observed between the time kinetics of the translocation of the enzyme. In plasma membranes of TPA-stimulated cells activation of protein kinase C was detectable up to 4 hr. In contrast, the highest specific activity of protein kinase C was measured in the plasma membranes after 15 min of DiC8 addition to cells. Anti-CD3 monoclonal antibodies activated protein kinase C in a biphasic manner. Shortly after binding of BMA 030 to the T cell antigen receptor/CD3 complex the activity of protein kinase C was increased in the plasma membrane, then it declined to control levels followed by a second long-lasting activation of the enzyme up to 4 hr. These results suggest different signal requirements for different activation steps. While for synthesis and expression of interleukin 2 receptors a short term activation of protein kinase C is sufficient, long-term activation of the enzyme is necessary for interleukin 2 synthesis in human lymphocytes.

Biological Transport↗