The P9 pocket of HLA-DQ2 (non-Aspbeta57) has no particular preference for negatively charged anchor residues found in other type 1 diabetes-predisposing non-Aspbeta57 MHC class II molecules

Susceptibility and resistance to type 1 diabetes are associated with MHC class II alleles that carry non-Asp and Asp at residue 57 of their beta chain respectively. The effect of Asp or non-Aspbeta57 may relate to a differential ability of distinct class II molecules to bind specific immuno-pathogenic peptides. Recent studies in man and mouse have revealed that some type 1 diabetes-predisposing non-Aspbeta57 class II molecules (i.e. DQ8, DR4Dw15 and I-Ag7) preferentially bind peptides with a negatively charged anchor residue at P9. It has been suggested that this is a common feature of type 1 diabetes-predisposing class II molecules. The molecular explanation for such a phenomenon could be that class II beta chains with Aspbeta57 form a salt bridge between Aspbeta57 and a conserved Arg of the a chain, whereas in non-Aspbeta57 molecules the Arg is unopposed and free to interact with negatively charged P9 peptide anchor residues. We have investigated the specificity of the P9 pocket of the type 1 diabetes-associated DQ2 molecule and in particular examined for charge effects at this anchor position. Different approaches were undertaken. We analyzed binding of a high-affinity binding ligand and P9-substituted variants of this peptide, and we analyzed the binding of a set of synthetic random peptide libraries. The binding analyses were performed with wild-type DQ2 and a mutated DQ2 with Ala at beta57 substituted with Asp. Our results indicate that the wild-type DQ2 (non-Aspbeta57) prefers large hydrophobic residues at P9 and that there is no particular preference for binding peptides with negatively charged residues at this position. The specificity of the P9 pocket in the mutated DQ molecule is altered, indicating that the beta57 residue contributes to determining the specificity of the P9 pocket. Our data do not lend support to the hypothesis that all non-Asp beta57 class II molecules predispose to development of disease by binding peptides with negatively charged P9 anchor residues.     

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.     

Synthesis of 2-isoxazoline and alpha-hydroxy ketomethylene dipeptide isosteres

We have developed a simple and stereoselective method for synthesizing novel dipeptide isosteres using nitrile oxide cycloaddition as a key reaction. Employing this method, we have prepared efficiently various peptidomimetics containing 2-isoxazolines and alpha-hydroxy ketomethylene dipeptide isosteres.     

Congenital dislocation of the patella

In 8 congenital patellar dislocations, in 7 patients, the patella was permanently and laterally dislocated, and irreducible or difficult to reduce. The patella usually was hypoplastic and showed absent facets. The intercondylar groove of the femur also was underdeveloped. In all but one patient, knee function was improved by a surgical procudure which involved lateral release of ptella, medial transpositon of patellar tendon, and plication of medial capsule or advancement of vastus medialis.     

Differential regulation of hepatic arterial and portal venous vascular resistance by nitric oxide and carbon monoxide in rats

Nitric oxide (NO), a gaseous mediator that accounts for the biological activity of endothelium-derived relaxing factor, has been shown to play an important role in the reduction of basal vascular tone in multiple vascular beds, including the hepatic circulation. On the other hand, recent studies have provided first evidence that endogenously generated carbon monoxide (CO) may exert vasodilatory effects in the hepatic portal vein and within sinusoids. Thus, we defined the differential role of NO and CO in the regulation of vascular resistance in the two inflows to the liver in the normal rat in vivo. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium and surgically instrumented in order to study the change in hepatic arterial (Rha) and portal venous vascular resistance (Rpv) in response to intravenous bolus administration of either the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mg/kg; n = 7 animals) or of tin protoporphyrin-IX (SnPP-IX) (50 micromol/kg), a specific inhibitor of the CO-generating enzyme heme oxygenase (n = 8 animals). While L-NAME caused a substantial increase in Rha, Rpv increased only slightly under these conditions. In sharp contrast, SnPP-IX did not affect Rha, but caused a profound increase in Rpv. In conclusion, Rha and Rpv are differentially regulated by NO and CO in the normal rat liver in vivo, i.e., NO serves as a potent vasodilator in the hepatic arterial circulation, but exerts only a minor vasodilatory effect in the portal venous vascular bed. In contrast, while there is no intrinsic CO-mediated vasodilation in the hepatic artery, CO acts to maintain portal venous vascular tone in a relaxed state.     

Diagnosing left lower lobe pneumonia: usefulness of the ''spine sign'' on lateral chest radiographs

Percutaneous cardiopulmonary assist devices (PCPS) have become available in interventional cardiology within recent years. These tools offer the opportunity of performing percutaneous transluminal coronary angioplasty (PTCA) in high-risk patients characterized by significant stenoses of several coronary arteries and a poor left ventricular function. It is unclear for which patients PCPS are necessary and which patients will profit by PTCA as compared to coronary artery bypass grafting (CABG). Therefore, the anticipated risk of CABG and of PTCA without assist devices was calculated according to risk scores and compared with our results of assisted PTCA. In addition the long-term survival rate was investigated. In 35 patients (mean 65.5 years of age, 12 females, 23 males), we performed PTCA concomitant with the use of cardiac assist devices. The indications for the use of a cardiac assist device were severely impaired LV function (EF 30% +/- 8.9%) in combination with significant coronary artery disease (2.7 +/- 0.3 vessels) and a significant supply area of the vessel to be dilated. In 6 patients, PCPS was started before coronary angioplasty because of hemodynamic instability. In 21 cases, PCPS was on a standby basis without being connected to the patient's circulation. In 8 patients, a left heart assist device, the 14F-Hemopump, was inserted percutaneously. The patients were analyzed using risk scores of angioplasty and of coronary bypass graft surgery. The calculated risk of hemodynamic compromise during PTCA according to the risk scores was more than 50%. The anticipated risk of a fatal outcome following CABG would have been 19.8%. PTCA was performed on an average of 2.0 coronary arteries per patient and was successful in 85%. We observed a decline in angina pectoris classification (CCS) from 3.5 to 1.6. An average reduction of 1.1 NYHA class was achieved. The in-hospital mortality was 8.6% (3 patients: 1 x sepsis, 1 x early reocclusion, 1 x cerebral embolism). At 24 months follow-up, a re-PTCA was necessary in four cases because of restenosis. In the remainder, NYHA and CCS class were stable during the follow-up period. An additional five patients died during the first year and two patients in the second year. We conclude that PTCA with the use of a cardiac assist device shows favorable short-term results in a subset of patients with extended coronary artery disease and severely impaired LV function who are not suitable for nonsupported PTCA or CABG due to their risk profile. However, the long term results are not satisfying and stress the need for complete revascularisation with CABG once the patient's condition is stabilized by means of supported PTCA.