Identification of the benzodiazepines as a new class of antileishmanial agent

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.     

A phosphatase activity of Sts-1 contributes to the suppression of TCR signaling

Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The X-ray structure of the Sts-1 C terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling.     

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.     

Function and stability of abscisic acid acyl hydrazone conjugates by LC-MS2 of ex vivo samples

We prepare a biotinylated conjugate of the ubiquitous plant hormone (S)-(+)-abscisic acid via an acyl hydrazone linkage at the C4' position and demonstrate in vivo cleavage of the otherwise stable acyl hydrazone linkage using LC-MS2. As part of a wider chemical genomic study, biological activity of the conjugate was assessed using standard epidermal peel and gravimetric transpiration assays, showing significant activity but at a level lower than the unconjugated hormone. When deuterated samples of the conjugate were fed to the plant, however, it was apparent by LC-MS2 experiments that significant levels of hydrolysis of the acyl hydrazone had taken place, contrary to in vitro stability assays in artificial sap. We conclude that abscisic acid is liberated in sufficient quantities to account for the observed physiological response and that LC-MS2 monitoring of conjugates is a simple and practical method by which such events may be assessed, whether in plants or other organisms.     

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.     

Experimental design criteria in phylogenetics: where to add taxa

Accurate phylogenetic inference is a topic of intensive research and debate and has been studied in response to many different factors: for example, differences in the method of reconstruction, the shape of the underlying tree, the substitution model, and varying quantities and types of data. Investigating whether the conditions used might lead to inaccurate inference has been attempted through elaborate data exploration but less attention has been given to creating a unified methodology to enable experimental designs in phylogenetic analysis to be improved and so avoid suboptimal conditions. Experimental design has been part of the field of statistics since the seminal work of Fisher in the early 20th century and a large body of literature exists on how to design optimum experiments. Here we investigate the use of the Fisher information matrix to decide between candidate positions for adding a taxon to a fixed topology, and introduce a parameter transformation that permits comparison of these different designs. This extension to Goldman (1998. Proc. R. Soc. Lond. B. 265: 1779-1786) thus allows investigation of "where to add taxa" in a phylogeny. We compare three different measures of the total information for selecting the position to add a taxon to a tree. Our methods are illustrated by investigating the behavior of the three criteria when adding a branch to model trees, and by applying the different criteria to two biological examples: a simplified taxon-sampling problem in the balsaminoid Ericales and the phylogeny of seed plants.     

Recent oceanic long-distance dispersal and divergence in the amphi-Atlantic rain forest genus Renealmia L.f. (Zingiberaceae)

Renealmia L.f. (Zingiberaceae) is one of the few tropical plant genera with numerous species in both Africa and South America but not in Asia. Based on phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F DNA, Renealmia is shown to be monophyletic with high branch support. Low sequence divergence found in the two genome regions (ITS: 0-2.4%; trnL-F: 0-1.9%) suggests recent diversification within the genus. Molecular divergence age estimates give further support to the recent origin of the genus and show that Renealmia has attained its amphi-Atlantic distribution by an oceanic long-distance dispersal event from Africa to South America during the Miocene or Pliocene (15.8-2.7 My ago). Some support is found for the hypothesis that speciation in neotropical Renealmia was influenced by the Andean orogeny. Speciation has been approximately simultaneous on both sides of the Atlantic, but increased taxon sampling is required to compare the speciation rates between the New World and Old World tropics.     

Reliability and detecting change following short-term creatine supplementation: comparison of two-component body composition methods

The purpose of the present study was twofold: firstly, to assess the reliability of various body composition methods, and secondly, to determine the ability of the methods to estimate changes in fat-free mass (FFM) following creatine (Cr) supplementation. Fifty-five healthy male athletes (weight 78.3 +/- 10.3 kg, age 21 +/- 1 years) gave informed consent to participate in this study. Subjects' FFM was estimated by hydrostatic weighing (HW), air-displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), near-infrared spectroscopy (NIR), and anthropometric measurements (ANTHRO). Measurements were taken on 2 occasions separated by 7 days to assess the reliability of the methods. Following this, 30 subjects returned to the laboratory for an additional test day following 7 days of Cr supplementation (20 g.d(-1) Cr + 140 g.d(-1) dextrose) to assess each method's ability to detect acute changes in FFM. In terms of reliability, we found excellent test-retest correlations for all 5 methods, ranging from 0.983 to 0.998 (p < 0.001). The mean biases for the 5 methods were close to 0 (range -0.1 to 0.3 kg) and their 95% limits of agreement (LOAs) were within acceptable limits (HW = -1.1 to 1.7 kg; ADP = -1.1 to 1.2 kg; BIA = -1.0 to 1.0 kg; NIR = -1.4 to 1.4 kg); however, the 95% LOAs were slightly wider for ANTHRO (-2.4 to 2.6 kg). Following Cr supplementation there was a significant increase in body mass (from 77.9 +/- 10.1 kg to 78.9 +/- 10.3 kg, p = 0.000). In addition, all 5 body composition techniques detected the change in FFM to a similar degree (mean change: HW = 0.9 +/- 0.6 kg; ADP = 0.9 +/- 0.6 kg; BIA = 0.9 +/- 0.6 kg; NIR = 0.8 +/- 0.5 kg; ANTHRO = 1.0 +/- 0.7 kg; intraclass correlation coefficient = 0.962). We conclude that between-day differences in FFM estimation were within acceptable limits, with the possible exception of ANTHRO. In addition, all 5 methods provided similar measures of FFM change during acute Cr supplementation.     

Interactive sequences in the stress protein and molecular chaperone human alphaB crystallin recognize and modulate the assembly of filaments

Molecular chaperones including the small heat shock proteins, alphaB crystallin and sHSP27 participate in the assembly, disassembly, and reorganization of the cytoskeleton during cell development and differentiation. While alphaB crystallin and sHSP27 stabilize and modulate filament assembly and re-organization, the sequences and structural domains mediating interactions between these proteins and filaments are unknown. It is important to define these interactive domains in order to understand differential interactions between chaperones and stable or unfolding filaments and their function in the cellular stress response. Protein pin arrays identified sequences in human alphaB crystallin that selectively interacted with native or partially unfolded filament proteins desmin, glial-fibrillary acidic protein, and actin. Circular dichroism spectroscopy determined differences in the structure of these filaments at 23 and 45 degrees C. Seven alphaB crystallin sequences had stronger interactions with desmin and six sequences had stronger interactions with glial-fibrillary acidic protein at 23 degrees C than at 45 degrees C. The alphaB crystallin sequences (33)LESDLFPTSTSLSPFYLRPPSFLR(56) and (129)DPLTITSSLSSDGV(145) had the strongest interactions with actin at 23 degrees C, while (57)APSWFDTG(64), (111)HGFISREF(118), (145)VNGPRKQVSG(154), and (155)PERTIPITREEK(165) had the strongest interactions with actin at 45 degrees C. The actin interactive sequences of alphaB crystallin overlapped with previously identified alphaB crystallin chaperone sequences and were synthesized to evaluate their effect on the assembly and aggregation of actin. Full-length alphaB crystallin and the core domain chaperone sequence (131)LTITSSLSSDGV(143) promoted actin polymerization at 37 degrees C and inhibited depolymerization and aggregation at 50 degrees C. The results support the hypothesis that interactive domains in alphaB crystallin have multiple functions in stabilizing the cytoskeleton and protecting cytosolic proteins from unfolding.