Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.     

Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells

Glucagon-like peptide 1 (GLP-1) is an intestine-derived insulinotropic hormone that stimulates glucose-dependent insulin production and secretion from pancreatic beta-cells. Other recognized actions of GLP-1 are to suppress glucagon secretion and hepatic glucose output, delay gastric emptying, reduce food intake, and promote glucose disposal in peripheral tissues. All of these actions are potentially beneficial for the treatment of type 2 diabetes mellitus. Several GLP-1 agonists are in clinical trials for the treatment of diabetes. More recently, GLP-1 agonists have been shown to stimulate the growth and differentiation of pancreatic beta-cells, as well as to exert cytoprotective, antiapoptotic effects on beta-cells. Recent evidence indicates that GLP-1 agonists act on receptors on pancreas-derived stem/progenitor cells to prompt their differentiation into beta-cells. These new findings suggest an approach to create beta-cells in vitro by expanding stem/progenitor cells and then to convert them into beta-cells by treatment with GLP-1. Thus GLP-1 may be a means by which to create beta-cells ex vivo for transplantation into patients with insulinopenic type 1 diabetes and severe forms of type 2 diabetes.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Glucagon-like peptide-1 receptor and proglucagon expression in mouse skin

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone expressed by alternative post-translational processing of proglucagon in the intestines, endocrine pancreas, and brain. The multiple antidiabetogenic actions of GLP-1 include stimulation of the proliferation and differentiation of the insulin-producing beta cells in the pancreas. The GLP-1 receptor is widely distributed and has been identified in the endocrine pancreas, intestinal tract, brain, lung, kidney, and heart. Here we report the expression of the GLP-1 receptor and proglucagon in the skin of newborn mice located predominantly in the hair follicles, as well as in cultures of skin-derived cells that also express nestin, a marker of cultured cells that have dedifferentiated by epithelial to mesenchymal transition. In cultured skin cells, GLP-1 activates the MAPK/ERK signal transduction pathway, associated with cellular proliferation, differentiation, and cytoprotection. No evidence was found for the activation of cAMP or Ca2+ signaling pathways. Further, redifferentiation of cultured skin-derived cells by incubation in differentiation medium containing GLP-1 induced expression of the proinsulin-derived peptide, C-peptide. These findings suggest a possible paracrine/autocrine role for GLP-1 and its receptor in skin development and possibly also in folliculogenesis.     

Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.     

Insights into structural network responsible for oligomerization and activity of bacterial virulence regulator caseinolytic protease P (ClpP) protein

The barrel-shaped caseinolytic protease P (ClpP) is a main virulence regulator in the bacterial pathogen Staphylococcus aureus (SaClpP). It consists of two heptameric rings forming a homotetradecamer with an inner chamber that houses the 14 active sites. We recently showed that SaClpP is able to adopt a compressed, inactive conformation. We present here the 2.3 Å resolution structure of SaClpP in its closed, active conformation as well as the structure of the S98A mutant. Comprehensive mutational analysis aiming at destabilizing one or the other or both conformations was able to pinpoint key residues involved in this catalytic switch and in the heptamer-heptamer interaction. By probing the active site serine with a covalently modifying β-lactone probe, we could show that the tetradecameric organization is essential for a proper formation of the active site. Structural data suggest that a highly conserved hydrogen-bonding network links oligomerization to activity. A comparison of ClpP structures from different organisms provides suggestive evidence for the presence of a universal mechanism regulating ClpP activity in which binding of one subunit to the corresponding subunit on the other ring interface is necessary for the functional assembly of the catalytic triad and thus for protease function. This mechanism ensures controlled access to the active sites of a highly unspecific protease.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Biomarkers and perfusion – training-induced changes after stroke (BAPTISe): protocol of an observational study accompanying a randomized controlled trial

Background

Physical activity is believed to exert a beneficial effect on functional and cognitive rehabilitation of patients with stroke. Although studies have addressed the impact of physical exercise in cerebrovascular prevention and rehabilitation, the underlying mechanisms leading to improvement are poorly understood. Training-induced increase of cerebral perfusion is a possible mediating mechanism. Our exploratory study aims to investigate training-induced changes in blood biomarker levels and magnetic resonance imaging in patients with subacute ischemic stroke.

Methods/design

This biomarker-driven study uses an observational design to examine a subgroup of patients in the randomized, controlled PHYS-STROKE trial. In PHYS-STROKE, 215 patients with subacute stroke (hemorrhagic and ischemic) receive either 4 weeks of physical training (aerobic training, 5 times a week, for 50 minutes) or 4 weeks of relaxation sessions (5 times a week, for 50 minutes). A convenience sample of 100 of these patients with ischemic stroke will be included in BAPTISe and will receive magnetic resonance imaging (MRI) scans and an additional blood draw before and after the PHYS-STROKE intervention. Imaging scans will address parameters of cerebral perfusion, vessel size imaging, and microvessel density (the Q factor) to estimate the degree of neovascularization in the brain. Blood tests will determine several parameters of immunity, inflammation, endothelial function, and lipometabolism. Primary objective of this study is to evaluate differential changes in MRI and blood-derived biomarkers between groups. Other endpoints are next cerebrovascular events and functional status of the patient after the intervention and after 3 months assessed by functional scores, in particular walking speed and Barthel index (co-primary endpoints of PHYS-STROKE). Additionally, we will assess the association between functional outcomes and biomarkers including imaging results. For all endpoints we will compare changes between patients who received physical fitness training and patients who had relaxation sessions.

Discussion

This exploratory study will be the first to investigate the effects of physical fitness training in patients with ischemic stroke on MRI-based cerebral perfusion, pertinent blood biomarker levels, and functional outcome. The study may have an impact on current patient rehabilitation strategies and reveal important information about the roles of MRI and blood-derived biomarkers in ischemic stroke.

Trial registration

NCT01954797.

     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.     

Silanetriols as in vitro inhibitors for AChE

Three stable silanetriols with increasing steric protection of the silicon atom have been tested for inhibition of acetylcholinesterase (AChE). For all tested silanetriols we found reversible inhibition of the AChE activity at a 100 μM concentration. The highest inhibition rate was found for the sterically least hindered cyclohexylsilanetriol with 45% inhibition relative to galanthamine hydrobromide for which an IC₅₀ value of 121 ± 3 μM was determined as well. The cytotoxicity of the silanetriols used was found to be negligible at concentrations relevant for inhibition.