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A Lahm

Publications and source records attributed to A Lahm.

53 records · Page 3Linked to original sources
PubMed1994

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.

Interleukin-6 (IL-6) drives the sequential assembly of a receptor complex formed by the IL-6 receptor (IL-6R alpha) and the signal transducing subunit, gp130. A model of human IL-6 (hIL-6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore essential for IL-6 signal transduction, were subjected to site-directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL-6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL-6-R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.

Amino Acid Sequence↗
PubMed1993

Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.

Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.

Amino Acid Sequence↗
PubMed1992

The Rhizobium leguminosarum biovar phaseoli glnT gene, encoding glutamine synthetase III.

Plasmid pGE203 contains the Rhizobium leguminosarum biovar phaseoli glnT locus. Glutamine synthetase III (GSIII) was purified from a glutamine auxotrophic strain of Klebsiella pneumoniae carrying this plasmid. Sequencing of a 2.4-kb fragment containing the glnT locus reveals an open reading frame of 435 amino acids (aa), whose first eight aa are identical to those determined from pure GSIII by direct aa sequencing, thus confirming that glnT indeed codes for GSIII activity. The comparison of the GSIII aa sequence with the reported sequence of GSs from other organisms shows a significant degree of homology. Since the three-dimensional structure of GS from Salmonella typhimurium is known, a three-dimensional model of GSIII was built by homology.

Amino Acid Sequence↗
PubMed1992

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.

Base Composition↗
PubMed1992

[Possibilities of nuclear magnetic resonance tomography in diagnosis of osteochondrosis dissecans of the knee joint].

20 patients who suffered from osteochondrosis dissecans in the knee were examined by x-ray and MRI. Afterwards all joints were evaluated by arthroscopy. On plain x-ray the judgement of joint cartilage is not possible. With the use of MRI, the hyaline cartilage can be documented. Even without any visible changes on x-ray, MRI clearly reveals pathologic bone patterns. Comparing areas of sclerosis on x-ray with MRI changes, the real diameter of osteochondrosis dissecans can only be seen on MRI.

Arthroscopy↗
PubMed1991

DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex.

The structure of a complex between DNase I and d(GCGATCGC)2 has been solved by molecular replacement and refined to an R-factor of 0.174 for all data between 6 and 2 A resolution. The nicked octamer duplexes have lost a dinucleotide from the 3' ends of one strand and are hydrogen-bonded across a 2-fold axis to form a quasi-continuous double helix of 14 base-pairs. DNase I is bound in the minor groove of the B-type DNA duplex forming contacts in and along both sides of the minor groove extending over a total of six base-pairs. As a consequence of binding of DNase I to the DNA-substrate the minor groove opens by about 3 A and the duplex bends towards the major groove by about 20 degrees. Apart from these more global distortions the bound duplex also shows significant deviations in local geometry. A major cause for the observed perturbations in the DNA conformation seems to be the stacking type interaction of a tyrosine ring (Y76) with a deoxyribose. In contrast, the enzyme structure is nearly unchanged compared to free DNase I (0.49 A root-mean-square deviations for main-chain atoms) thus providing a rigid framework to which the DNA substrate has to adapt on binding. These results confirm the hypothesis that groove width and stiffness are major factors determining the global sequence dependence of the enzyme's cutting rates. The nicked octamer present in the crystals did not allow us to draw detailed conclusions about the catalytic mechanism but confirmed the location of the active site near H134 on top of the central beta-sheets. A second cut of the DNA induced by diffusion of Mn2+ into the crystals may suggest the presence of a secondary active site in DNase I.

Amino Acid Sequence↗
PubMed1991

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

Acetylglucosamine↗
PubMed1991

[Concomitant occult osseous injuries in knee joint injuries].

In 28 patients with different knee injuries, we documented intraosseous lesions by MRI, although on plain X-rays no abnormality had been discovered. The intraosseous abnormalities seen by MRI were classified into four categories. 1. Cancellous bone oedema: this type of lesion showed a decrease of signal on T1 weighted images with irregular borders. These irregular areas were located in the epiphysis and sometimes even in the metaphysis. On T2 weighted images these areas showed an increased signal, which was even better visualised on short time recovery sequences. 2. Subchondral fracture with involvement of cortical bone: In these cases the involved area even reached the subchondral cortex. The fracture line could be visualised on T1- and T2-weighted images. 3. Osteochondral fractures: In two patients we found typical osteochondral fractures with a minimal displaced small fragment. 4. Subchondral sclerosis: In these cases there was a decrease of the signal on T1-weighted images, located directly adjacent to the subchondral cortical bone, without a fracture within the cortical bone and without any increase of signal on T2-weighted or STIR-images. According to our experience, isolated decrease in the signal within the subchondral cancellous bone adjacent to the knee joint, with an increased signal in STIR sequences can be taken as indirect signs for a traumatic lesion. These intraosseous lesions have a high coincidence with ligament injuries of the knee joint.

Adolescent↗
PubMed1991

[Proton spin and computerized tomography in ligament injuries of the knee joint].

UNLABELLED: In a prospective study we evaluated the use of cat-scan an MRI in 106 patients with chronic complaints and acute injuries of the knee joint. All patients went to surgery afterwards. A total of 30 patients suffered from acute or chronic ligament ruptures. There were 20 ACL ruptures, 2 PCL ruptures, 3 MCL ruptures, and 2 LCL ruptures. In patients with ACL ruptures the MRI had a sensitivity of 80%, a specificity value of 80%, and a negative predictive value of 95%; whereas cat-scan had a sensitivity of 90%, a specificity of 95%, an accuracy of 94%, a positive predictive value of 82%, and a negative predictive value of 98%. All complete ACL ruptures we correctly diagnosed. However, 4 partial ACL ruptures were missed by MRI and 2 partial ruptures were missed by cat-scan. All other ligament ruptures (PCL, MCL, LCL) except one MCL rupture were correctly diagnosed by MRI as well as by cat-scan. CLINICAL RELEVANCE: In this study cat-scan and MRI proved to have a very high negative predictive value. Having a negative result in one of these imaging techniques means that a rupture of a ligament is highly unlikely. This may have influence on the number of unnecessary invasive arthroscopic procedures.

Anterior Cruciate Ligament Injuries↗
PubMed1991

[Nuclear magnetic resonance and computerized tomography in meniscus injuries of the knee joint].

In a prospective study we evaluated the use of CAT scan and MRI in 53 patients with chronic symptoms and acute injuries of the knee joint. All these patients subsequently underwent surgery. A total of 21 patients had acute or chronic meniscus ruptures. The sensitivity of MRI was 86% (medial: 88%, lateral: 75%), its specificity, 95% (medial: 92%, lateral: 98%), its accuracy 93% (medial: 91%, lateral: 96%), its positive predictive value of 82% (medial: 83%, lateral: 75%), and its negative predictive value 96% (medial: 94%, lateral: 98%); CAT scan had a sensitivity of 86% (medial: 88%, lateral: 75%), a specificity of 97% (medial: 94%, lateral: 98%), an accuracy of 94% (medial: 93%, lateral: 96%), a positive predictive value of 86% (medial: 88%, lateral: 75%), and a negative predictive value of 97% (medial: 94%, lateral: 98%). In this study CAT scan and MRI proved to have a very high negative predictive value in the diagnostic of meniscal lesions. A negative result with one of these imaging techniques means that a rupture of a meniscus is highly unlikely. This may avoid unnecessary invasive procedures such as arthrotomies or arthroscopic examinations.

Arthroscopy↗
PubMed1990

Crystallisation and preliminary crystallographic analysis of P1 nuclease from Penicillium citrinum.

P1 nuclease, a zinc-dependent single-strand specific endonuclease from Penicillium citrinum, has been crystallized in three different space groups using either ammonium sulphate or polyethylene glycol 4000 as the precipitating agent. The crystals diffract to between 3 A and 2.2 A. A 4.5 A electron density map has been calculated for a tetragonal crystal form, based on a platinum derivative, and was improved by solvent flattening. The boundaries of the two molecules in the asymmetric unit are clearly visible in most regions and the presence of rod-like density features are indicative of a rather high alpha-helix content. The highest density peaks in the map were identified as a trinuclear zinc cluster present in each monomer by a difference Fourier of an EDTA-soaked crystal.

Crystallography↗
PubMed1988

Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I.

The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.

Deoxyribonuclease I↗
PubMedDate not supplied

Structural conservation in single-domain proteins: implications for homology modeling.

Large-scale sequencing projects are widening the gap between the known protein universe and the fraction for which structural information has been experimentally obtained. Through the application of homology (comparative) modeling and more general structure prediction techniques, this gap can, however, be narrowed, providing indirect structural information for a considerable number of proteins. Moreover, the estimated number of existing protein folds seems to be limited and many of these yet unknown folds should be discovered by dedicated large-scale structural genomics projects. Within this perspective, homology (comparative) modeling will gain in importance, as will the use of models derived by this technique. Here we discuss how well a sequence alignment, the most common starting point for generating a model, reflects the structural conservation between homologous proteins and we show that sequence information is able to direct construction of acceptable models as far as the structural core is concerned. We also show here that the regions surrounding insertions and deletions are much less conserved than the core and discuss the implications of this observation for loop modeling.

Amino Acid Sequence↗
PubMedDate not supplied

[Intravenous administration of gadolinium DPTA in proton spin tomography of the knee joint].

UNLABELLED: In a prospective study we evaluated the benefits of iv-application of gadolinium-DPTA examining 25 patients with degenerative disease of the knee joint by MRI (1.0 Tesla). All patients were scanned with standard spin echo sequences (SE 500/20), short time inversion recovery sequences (STIR 1600/100), and FEDIF sequences (FEDIF 500/10). After gadolinium-DPTA application (0.1-0.35 mmol/kg BW) the patients were scanned with spin echo sequences (SE SE 500/20), and FEDIF sequences (FEDIF 500/10). After iv-Gadolinium the synovial membrane of joints with inflammation show significant increase of the signal. Due to this effect a differentiation between joint effusion and hypertrophic synovia is possible. Small areas of local synovitis not visible before the use of gadolinium can be documented. A differentiation between degenerative subchondral cysts and cysts due to an inflammatory process is possible. CLINICAL RELEVANCE: The use of iv-application of gadolinium-DPTA in patients with degenerative joint disease seems to offer additional information. With iv-application all shortcomings of intraarticular use can be avoided. The differentiation between degenerative and inflammatory processes is possible.

Contrast Media↗
PubMedDate not supplied

[The value of nuclear magnetic resonance tomography in diseases of the knee joint].

In a prospective study we examined 107 patients suffering from an acute trauma or a chronic disease of the knee joint by MRI. The MRI results were compared to the results of arthroscopy. Normal anatomical structures can be differentiated and there is a high diagnostic reliability for lesions of the medial (accuracy: 91%, predictive value: 84%) and lateral (accuracy: 93%, predictive value: 79%) meniscus, complete tears of the anterior cruciate ligament (accuracy: 93%, predictive value: 75%), chondropathia of the patella, and osteochondritis dissecans. Partial ruptures of the ACL as well as small cartilage defects could usually not be demonstrated.

Arthritis, Rheumatoid↗