Cloning, purification and characterization of a functional anthracycline glycosyltransferase

We have cloned the gene that encodes a novel glucosyl transferase (AraGT) involved in rhamnosylation of the polyketide antibiotic Aranciamycin in Streptomyces echinatus. AraGT comprises two domains characteristic of bacterial glycosyltranferases. AraGT was synthesized in E. coli as a decahistidinyl-tagged polypeptide. Purified AraGT is dimeric, displays a T(mapp) of 30 degrees C and can glycosylate the aglycone of an Aranciamycin derivative as shown by liquid chromatography and mass spectrometry. The availability of functional AraGT will allow the generation Aranciamycin-based combinatorial libraries.     

Role of gamma-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.     

Maraviroc, a chemokine receptor-5 antagonist, fails to demonstrate efficacy in the treatment of patients with rheumatoid arthritis in a randomized, double-blind placebo-controlled trial

Introduction

The purpose of this study was to determine whether maraviroc, a human CC chemokine receptor 5 (CCR5) antagonist, is safe and effective in the treatment of active rheumatoid arthritis (RA) in patients on background methotrexate (MTX).

Methods

This phase IIa study comprised two distinct components: an open-label safety study of the pharmacokinetics (PK) of MTX in the presence of maraviroc, and a randomized, double-blind, placebo-controlled, proof-of-concept (POC) component. In the PK component, patients were randomized 1:1 to receive maraviroc 150 or 300 mg twice daily (BID) for four weeks. In the POC component, patients were randomized 2:1 to receive maraviroc 300 mg BID or placebo for 12 weeks. Patients were not eligible for inclusion in both components.

Results

Sixteen patients were treated in the safety/PK component. Maraviroc was well tolerated and there was no evidence of drug-drug interaction with MTX. One hundred ten patients were treated in the POC component. The study was terminated after the planned interim futility analysis due to lack of efficacy, at which time 59 patients (38 maraviroc; 21 placebo) had completed their week 12 visit. There was no significant difference in the number of ACR20 responders between the maraviroc (23.7%) and placebo (23.8%) groups (treatment difference -0.13%; 90% CI -20.45, 17.70; P = 0.504). The most common all-causality treatment-emergent adverse events in the maraviroc group were constipation (7.8%), nausea (5.2%), and fatigue (3.9%).

Conclusions

Maraviroc was generally well tolerated over 12 weeks; however, selective antagonism of CCR5 with maraviroc 300 mg BID failed to improve signs and symptoms in patients with active RA on background MTX.

Trial Registration

ClinicalTrials.gov: NCT00427934     

The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum

The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid.     

Coarse-grained molecular simulation of diffusion and reaction kinetics in a crowded virtual cytoplasm

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.     

Therapeutic role of sirtuins in neurodegenerative disease

The sirtuins are a family of enzymes which control diverse and vital cellular functions, including metabolism and aging. Manipulations of sirtuin activities cause activation of anti-apoptotic, anti-inflammatory, anti-stress responses, and the modulation of an aggregation of proteins involved in neurodegenerative disorders. Recently, sirtuins were found to be disease-modifiers in various models of neurodegeneration. However, almost in all instances, the exact mechanisms of neuroprotection remain elusive. Nevertheless, the manipulation of sirtuin activities is appealing as a novel therapeutic strategy for the treatment of currently fatal human disorders such as Alzheimer's and Parkinson's diseases. Here, we review current data which support the putative therapeutic roles of sirtuin in aging and in neurodegenerative diseases and the feasibility of the development of sirtuin-based therapies.     

Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.     

On the use of partial area under the ROC curve for comparison of two diagnostic tests

Evaluation of diagnostic performance is typically based on the receiver operating characteristic (ROC) curve and the area under the curve (AUC) as its summary index. The partial area under the curve (pAUC) is an alternative index focusing on the range of practical/clinical relevance. One of the problems preventing more frequent use of the pAUC is the perceived loss of efficiency in cases of noncrossing ROC curves. In this paper, we investigated statistical properties of comparisons of two correlated pAUCs. We demonstrated that outside of the classic model there are practically reasonable ROC types for which comparisons of noncrossing concave curves would be more powerful when based on a part of the curve rather than the entire curve. We argue that this phenomenon stems in part from the exclusion of noninformative parts of the ROC curves that resemble straight‐lines. We conducted extensive simulation studies in families of binormal, straight‐line, and bigamma ROC curves. We demonstrated that comparison of pAUCs is statistically more powerful than comparison of full AUCs when ROC curves are close to a “straight line”. For less flat binormal ROC curves an increase in the integration range often leads to a disproportional increase in pAUCs’ difference, thereby contributing to an increase in statistical power. Thus, efficiency of differences in pAUCs of noncrossing ROC curves depends on the shape of the curves, and for families of ROC curves that are nearly straight‐line shaped, such as bigamma ROC curves, there are multiple practical scenarios in which comparisons of pAUCs are preferable.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.