GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex

Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser¹⁶³ and Ser¹⁶⁷. The importance of these phosphorylation events was determined by mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser¹⁶³ and Ser¹⁶⁷ in GAP45 controls the final step in assembly of the myosin XIV motor complex.     

Remission in psoriatic arthritis: is it possible and how can it be predicted?

Introduction

Since remission is now possible in psoriatic arthritis (PsA) we wished to examine remission rates in PsA patients following anti tumour necrosis factor alpha (TNFα) therapy and to examine possible predictors of response.

Methods

Analysis of a prospective patient cohort attending a biologic clinic, between November 2004 and March 2008, was performed prior to commencing therapy and at regular intervals. Baseline clinical characteristics including demographics, previous disease-modifying antirheumatic drug (DMARD) response, tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and health assessment questionnaire (HAQ) were collected.

Results

A total of 473 patients (152 PsA; 321 rheumatoid arthritis (RA)) were analyzed. At 12 months remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients compared to 44% of RA patients, significant improvement in outcome measures were noted in both groups (P < 0.05). Analysis of a subgroup of PsA and RA patients matched for DAS28-CRP at baseline also showed higher numbers of PsA patients achieving remission. Linear regression analysis identified the HAQ at baseline as the best predictor of remission in PsA patients (P < 0.001).

Conclusions

DAS28 remission is possible in PsA patients at one year following anti-TNF therapy, at higher rates than in RA patients and is predicted by baseline HAQ.     

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed

Background

Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed.

Methods

46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity.

Results

Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, R_t = 5.7 ± 1.4, H_o = 0.63 ± 0.19 and H_e = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (F_is = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (F_st = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity.

Conclusions

Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.     

A review of recent non-target toxicity testing of vertebrate pesticides: establishing generic guidelines

Abstract

An analysis of the range, extent and importance of New Zealand-based ecotoxicology studies has been completed to better understand the hazards and non-target risks associated with new toxins and baits. This review focuses on compounds that have been recently developed for incorporation into bait for terrestrial vertebrate pest control, namely cholecalciferol, para-aminopropiophenone (PAPP) and zinc phosphide and the effects of these toxins on non-target species. Testing of these compounds has included cage and pen toxicity and bait acceptance studies. Locally conducted acute toxicity studies clarify overseas data and enhance risk assessments. Consistency in approach and selection of surrogate species, and careful selection of native non-target species for acute toxicity testing in cage and pen trials are recommended as important steps before field trials. We propose a systematic approach to cage and pen trials and offer guidelines on species selection.     

Analysis of the hydrogenotrophic microbiota of wild and captive black howler monkeys (Alouatta pigra) in palenque national park, Mexico

Intestinal methanogenesis is one of the major pathways for consumption of hydrogen produced by bacterial fermentation and is considered to affect the efficiency of host energy harvest; however, little information is available regarding the hydrogenotrophic pathways of nonhuman primates in the wild, in general, and of howler monkeys, in particular. Microbial fermentation of plant structural carbohydrates is an important feature in wild howlers owing to the high fiber and low available energy content of leaves, which make up the primary component of their diet. In contrast, captive howlers may consume greater quantities of fruits and vegetables that are higher in water, lower in fiber, and, along with commercial monkey chow commonly added to captive monkey diets, more readily digestible than the natural diet. In this study, we analyzed the composition of methanogens and sulfate-reducing bacteria (SRB) from fecal samples of black howler monkeys (Alouatta pigra) in the wild and in captivity. The hydrogenotrophic microbiota of three groups of monkeys was evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, small clone library construction, and quantitative real-time PCR. Abundance of methanogens was lower than SRB in all howler monkey groups studied. DGGE banding patterns were highly similar within each wild and captive group but distinct among groups. Desulfovibrionales-enriched DGGE showed reduced microbial diversity in the captive animals compared with their wild counterparts. Taken together, the data demonstrate that environmental or dietary changes of the host imposed by captivity likely influence the composition of intestinal hydrogenotrophs in black howler monkeys.     

Examining Effects of Anticipated Stigma,Centrality, Salience,Internalization, and Outness on Psychological Distress for People with Concealable Stigmatized Identities

Understanding how stigmatized identities contribute to increased rates of depression and anxiety is critical to stigma reduction and mental health treatment. There has been little research testing multiple aspects of stigmatized identities simultaneously. In the current study, we collected data from a diverse, urban, adult community sample of people with a concealed stigmatized identity (CSI). We targeted 5 specific CSIs – mental illness, substance abuse, experience of domestic violence, experience of sexual assault, and experience of childhood abuse – that have been shown to put people at risk for increased psychological distress. We collected measures of the anticipation of being devalued by others if the identity became known (anticipated stigma), the level of defining oneself by the stigmatized identity (centrality), the frequency of thinking about the identity (salience), the extent of agreement with negative stereotypes about the identity (internalized stigma), and extent to which other people currently know about the identity (outness). Results showed that greater anticipated stigma, greater identity salience, and lower levels of outness each uniquely and significantly predicted variance in increased psychological distress (a composite of depression and anxiety). In examining communalities and differences across the five identities, we found that mean levels of the stigma variables differed across the identities, with people with substance abuse and mental illness reporting greater anticipated and internalized stigma. However, the prediction pattern of the variables for psychological distress was similar across the substance abuse, mental illness, domestic violence, and childhood abuse identities (but not sexual assault). Understanding which components of stigmatized identities predict distress can lead to more effective treatment for people experiencing psychological distress.     

Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors

The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with F?rster resonance energy transfer (FRET). Alpha-helical linkers differing in length were combined with motifs that are sensitive to the redox state of the environment. The first category of linkers included a redox motif found in the thioredoxin family of oxidoreductases. This motif was flanked by two alpha-helices of equal length. The second and third categories of redox linkers were composed of alpha-helices with embedded adjacent and dispersed vicinal cysteine residues, respectively. The linkers containing redox switches were placed between a FRET pair of enhanced cyan and yellow fluorescent proteins and these constructs were tested subsequently for their efficacy. A robust method of FRET analysis, the (ratio)(A) method, was used. This method uses two fluorescence spectra performed directly on the FRET construct without physical separation of the fluorophores. The cyan/yellow construct carrying one of the designed redox linkers, RL5, exhibited a 92% increase in FRET efficiency from its reduced to oxidized states. Responsiveness of the cyan-RL5-yellow construct to changes in the intracellular redox environment was confirmed in mammalian cells by flow cytometry.     

Antibiotics as growth promotants: mode of action

Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.     

Proteolytic processing of TgIMC1 during maturation of the membrane skeleton of Toxoplasma gondii

Membrane skeletons play an important role in the maintenance of cell shape and integrity in many cell types. In the protozoan parasite Toxoplasma gondii this function is performed by the subpellicular network, a resilient structure composed of tightly interwoven 10-nm filaments. We report here that this network is assembled at an early stage in the development of daughter parasites. The networks of immature and mature parasites differ dramatically with respect to their stability. Although in immature parasites the network is completely solubilized by detergent, the network in mature parasites is entirely detergent-resistant. Conversion of the detergent-labile to the detergent-resistant network occurs late in daughter cell development and appears to be coupled to proteolytic processing of the carboxyl terminus of TgIMC1, the major subunit of the network filaments. A single cysteine residue in the TgIMC1 carboxyl terminus was found to be essential for this processing event. The dramatic change in resistance to detergent extraction probably reflects an overall change in structural stability of the subpellicular network that accompanies maturation of daughter parasites and allows a switch from an assembly-competent but loose structure to one that is rigid and offers mechanical strength to the mature parasite.