Crystallization of YIoQ, a GTPase of unknown function essential for Bacillus subtilis viability

YLoQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 Angstrom spacing

The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7 angstrom resolution

Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5' and 3' ends. Cleavage of nucleotides from the 3' stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B. anthracis has been solved by molecular replacement to a resolution of 1.7 angstrom and refined to an R factor of 19.3%. There is one molecule in the asymmetric unit; the crystal packing reveals the assembly of the protein into a hexamer arranged as a trimer of dimers. The structure shows two sulfate ions bound in the active-site pocket, probably mimicking the phosphate substrate and the phosphate of the 3'-terminal nucleotide of the tRNA precursor. Three other bound sulfate ions point to likely RNA-binding sites

Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilis

AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholino-ethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3 Angstrom spacing has been collected

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

Long-term Results of Drug and Interventional Treatment in Patients with Morphologically Verified Idiopathic Arrhythmias

Aim. To study the late results of medical and interventional treatment in patients with morphologically verified nature of idiopathic arrhythmias.Methods. The prospective study included 20 patients (mean age 43.1±11.3 years, 10 female) with atrial fibrillation (AF), supraventricular and ventricular extrasystole, supraventricular and ventricular tachycardia, conduction disturbance without structural heart changes. In addition to the standard examination, the level of anti-heart antibodies was initially determined; endomyocardial biopsy (EMB) of the right ventricle with PCR study for the viral genome; DNA diagnostics (n=4), coronary angioraphy (n=6), skin biopsy (n=1) were performed. The median follow-up was 134 [128; 138] months.Results. By EMB in the initial examination were diagnosed: active (n=8)/borderline (n=3) infectious immune myocarditis; parvovirus-positive endomyocarditis (n=1); undifferentiated vasculitis (n=2); myocardial vasculitis (n=1); Fabry disease (n=1); arrhythmogenic right ventricular dysplasia (n=1); unspecified cardiomyopathy (n=2). Anti-heart antibodies were the most important in myocarditis diagnosis and monitoring. All patients with myocarditis/vasculitis (n=15) received its basic therapy: acyclovir (n=10); immunoglobulin G 10-12.5 g (n=2); hydroxychloroquine 200 mg/day (n=15); glucocorticoids (n=14); azathioprine 150 mg/day (n=2). The late results were evaluated in all patients with myocarditis. Initially, in 62.5% of patients a resistance of AF to all antiarrhythmic drugs was noted. After treatment the average frequency of AF paroxysms decreased (from 8 [5; 8] to 3 [1,25; 7,75] points). By the end of the follow-up, six patients underwent radiofrequency ablation (RFA) for AF, the full effect was achieved once. All patients without RFA have AF partially or completely resistant to drugs. Two patients (without RFA) died from ischemic stroke/ pulmonary embolism.Conclusion. Using EMB the causes of idiopathic arrhythmias (mainly AF) were diagnosed: immune inflammatory diseases in 75% and genetic in 25% of patients. As a result of complex treatment, the general burden of arrhythmias has decreased. But the presence of myocarditis and primary cardiomyopathy, without reducing the cardiac contractility and dilatation, does not allow achieving a stable antiarrhythmic effect. Lethality for 11 years was 10%. The causes of death were thromboembolic complications

Crystal growth and preliminary X-ray study of glutamic acid specific serine protease from Bacillus intermedius

The glutamic acid specific protease (glutamyl-endopeptidase) from Bacillus intermedius, strain 3-19, was isolated and purified using ion exchange chromatography on CM-cellulose and Mono-S FPLC column. The conditions for crystallization of the enzyme have been discussed. The crystals of enzyme were grown using hanging-drop vapor-diffusion technique. Crystals belong to the space group C2 with unit cell parameters of a = 61.62 Å, b = 55.84 Å, c = 60.40 Å, β = 117.6° X-ray diffraction data to 1.68 Å resolution were collected using synchrotron radiation (EMBL, Hamburg) and an imaging plate scanner. © 1999 Elsevier Science B.V. All rights reserved

Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase v

α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation

Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity

Rendu-Osler-Weber Disease with High Pulmonary Hypertension and Interstitial Lung Disease

A 64-year-old female with a family history of hereditary hemorrhagic telangiectasia (HHT) was hospitalized due to complaints of dyspnea during light physical exertion and leg edema. HHT was diagnosed at 20 y.o., recurrent nasal bleeding started at age 52, bleedings severity was aggravated by not completely compensated hypertension. At the age of 60, after a massive hemorrhage, she noted the onset of dyspnea, edema, ascites. Diuretics and iron preparations improved her well-being, but from that period onward her heart failure worsened after each massive blood loss. The last major bleeding was before the present hospitalization (Hgb 67 g/l), after which heart failure symptoms significantly deteriorated. Echocardiography showed preserved left ventricular ejection fraction, but revealed high pulmonary hypertension (systolic pulmonary artery pressure 69 mmHg). Chest computed tomography (CT) with contrast showed no evidence of pulmonary embolism, but interstitial lung lesions were detected. Pulse therapy with glucocorticosteroids did not result in positive dynamics at the control CT scan, which allowed to reject a separate interstitial lung disease. As a result of cardiotropic and diuretic therapy, as well as correction of anemia, the patient's condition improved. Macitentan was administered, but the patient refused from it because one of possible side effects was anemia. A year later the patient diedfrom acute progression of pulmonary hypertension. According to the literature, pulmonary hypertension in HHT can have a significant impact on the prognosis and requires timely diagnosis and treatment. Interstitial lung lesions are a manifestation of the underlying disease and does not require special treatment