A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Genetic dissection of gene regulation in multiple mouse tissues

The analysis of the influence of genetic variation on regulation of gene expression at a near-genome-wide level has become the focus of much recent interest. It is widely appreciated that many genes are expressed in a tissue-specific manner and that others are more ubiquitously expressed but relatively little is known about how genetic variation might influence these tissue patterns of gene expression. In this review we discuss what is known about the tissue specificity of the influence of genetic variation and review the challenges that we face in combining hugely parallel, microarray-based gene analysis with equally expensive genetic analysis. We conclude that the available data suggest that genetic variation is essentially tissue specific in its effects upon gene expression and this has important implications for experimental analysis.     

Glenohumeral elevation-dependent influence of anterior glenohumeral capsular lesions on passive axial humeral rotation

The purpose of this study was to assess the effect of standardized anterior glenohumeral capsular lesions on axial humeral rotation in a full arc of glenohumeral elevation. Using a testing apparatus, the range of internal and external humeral rotation was assessed in an arc of glenohumeral elevation in the scapular plane with steps of 15 degrees in six isolated shoulder joint specimens. Cutting of the glenohumeral joint capsule 1 cm laterally from, and parallel to the glenoid rim was performed in seven steps of 1 cm till the anterior capsule was cut. Capsular lesions were made in three ways: from inferior, from superior and from the middle of the capsule. Anterior capsular lesions resulted in significant increase of external humeral rotation. This occurred particularly at 15-60 degrees glenohumeral elevation. Lesions of the inferior part of the capsule mainly increased external rotation at 30-60 degrees glenohumeral elevation, lesions of the superior part mainly in lower elevation angles and lesions of the middle more gradually in the range till 60 degrees of glenohumeral elevation. Cutting of the anterior glenohumeral capsule barely increased passive axial humeral rotation at elevation angles over 60 degrees. Above 60 degrees glenohumeral elevation, tightening of the inferior posterior glenohumeral joint capsule prevented both internal and, increasingly, external humeral rotation. From these observations it is concluded that increased external rotation correlates with progressive anterior capsular lesions, mainly below 60 degrees glenohumeral elevation. To assess anterior glenohumeral capsular lesions in patients, axial humeral rotation tests should probably not exceed 60 degrees glenohumeral elevation, i.e. 90 degrees thoracohumeral elevation.     

Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.     

Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.     

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.