Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II

The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.     

Comparative phylogeography of six herpetofauna species in Cyprus: late Miocene to Pleistocene colonization routes

The colonization patterns of oceanic islands are often interpreted through transmarine dispersal. However, in islands with intense human activities and unclear geological history, this inference may be inappropriate. Cyprus is such an island, whose geotectonic evolution has not been clarified yet to the desired level for biogeographical reconstructions, leaving the questions of ‘how the Cypriote biota arrived’ and ‘does the dispersal have the formative role in patterns of its diversification’ unanswered. Here, we address these issues through a reconstruction of the evolutionary history of six herptiles (Ablepharus budaki, Ophisops elegans, Acanthodactylus schreiberi, Telescopus fallax, Pelophylax cf. bedriagae, and Hyla savignyi) by means of mitochondrial DNA (cytochrome b and 16S rRNA), applying a Bayesian phylogenetic, biogeographical, and chronophylogenetic analyses. The phylogeographical analyses show that the colonization history of those species in Cyprus started in the late Miocene and extended into the Pliocene and Pleistocene, with geodispersal, transmarine dispersal, and human‐mediated dispersal having their share in shaping the diversification of Cypriote herptiles. The revealed patterns could be divided into three biogeographical categories: old colonizers that arrived in Cyprus during the late Miocene or early Pliocene either by a land bridge (geodispersal) which connected Cyprus with the mainland or by transmarine dispersal, younger colonizers that reached the island through transmarine dispersal from the Middle East, and new settlers that arrived through human‐induced (voluntary or not) introductions. This work advances our knowledge of the biogeography of Cyprus and highlights the need to consider both geo‐ and transmarine dispersal when dealing with islands whose associations do not have a straightforward interpretation. © 2013 The Linnean Society of London     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Victims and survivors: Stable isotopes used to identify migrants from the Great Irish Famine to 19th century London

Historical evidence documents mass migration from Ireland to London during the period of the Great Irish Famine of 1845–52. The rural Irish were reliant on a restricted diet based on potatoes but maize, a C₄ plant, was imported from the United States of America in 1846–47 to mitigate against Famine. In London, Irish migrants joined a population with a more varied diet. To investigate and characterize their diet, carbon and nitrogen isotope ratios were obtained from bone collagen of 119 and hair keratin of six individuals from Lukin Street cemetery, Tower Hamlets (1843–54), and bone collagen of 20 individuals from the cemetery at Kilkenny Union Workhouse in Ireland (1847–51). A comparison of the results with other contemporaneous English populations suggests that Londoners may have elevated δ¹⁵N compared with their contemporaries in other cities. In comparison, the Irish group have lower δ¹⁵N. Hair analysis combined with bone collagen allows the reconstruction of perimortem dietary changes. Three children aged 5–15 years from Kilkenny have bone collagen δ¹³C values that indicate consumption of maize (C₄). As maize was only imported into Ireland in quantity from late 1846 and 1847, these results demonstrate relatively rapid bone collagen turnover in children and highlight the importance of age‐related bone turnover rates, and the impact the age of the individual can have on studies of short‐term dietary change or recent migration. Stable light isotope data in this study are consistent with the epigraphic and documentary evidence for the presence of migrants within the London cemetery. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Novel 3-hydroxy vinylboronates influence sphingolipid metabolism,cause apoptosis in Jurkat cells and prevent tumor development in nude mice

A series of novel 3-hydroxy vinylboronates which share structural similarities with sphingolipids were synthesized and tested in vitro and in vivo as anticancer agents. The molecules reduced cancer cell survival in vitro by influencing their sphingolipid metabolism. In a cancer model in nude mice the lead compound E7 prevented the development of tumor as long as the treatment period continued. Moreover, it delayed tumor growth after the treatment was finished.     

Phylogenetic relationships of the populations of Iranolacerta brandtii (de Filippi, 1863) (Squamata: Lacertidae) recently found in Eastern Anatolia,Turkey

The Persian Lizard, Iranolacerta brandtii, was until recently considered to be restricted to north-western Iran (Azerbaijan and Esfahan provinces). However, two recent studies have revealed the existence of populations in Eastern Anatolia, extending the range of this species for about 230?km westwards. The fragmented distribution of this species has been considered to be a consequence of the climatic oscillations during the Pleistocene and Holocene, which created events of alternating contact and isolation of populations in distinct glacial refugia. According to our obtained genealogy derived from three mitochondrial fragments (12S rRNA, 16S rRNA and cytb), the Turkish specimens cluster together but form an independent clade, sister to the individuals from Tabriz in Iran. The separation of these two clades is concurrent with the cladogenesis between the Esfahan and Ardabil clades, estimated to have taken place during the late Holocene.     

Maternal Conditions and Perinatal Characteristics Associated with Autism Spectrum Disorder and Intellectual Disability

Background

As well as being highly comorbid conditions, autism spectrum disorders (ASD) and intellectual disability (ID) share a number of clinically-relevant phenomena. This raises questions about similarities and overlap in diagnosis and aetiological pathways that may exist for both conditions.

Aims

To examine maternal conditions and perinatal factors for children diagnosed with an ASD, with or without ID, and children with ID of unknown cause, compared with unaffected children.

Methods

The study population comprised all live singleton births in Western Australia (WA) between January 1984 and December 1999 (N = 383,153). Univariate and multivariate multinomial logistic regression models were applied using a blocked modelling approach to assess the effect of maternal conditions, sociodemographic factors, labour and delivery characteristics and neonatal outcomes.

Results

In univariate analyses mild-moderate ID was associated with pregnancy hypertension, asthma, urinary tract infection, some types of ante-partum haemorrhage, any type of preterm birth, elective C-sections, breech presentation, poor fetal growth and need for resuscitation at birth, with all factors showing an increased risk. Severe ID was positively associated with poor fetal growth and need for resuscitation, as well as any labour or delivery complication. In the multivariate analysis no maternal conditions or perinatal factors were associated with an increased risk of ASD without ID. However, pregnancy hypertension and small head circumference were associated with a reduced risk (OR = 0.64, 95% CI: 0.43, 0.94; OR = 0.58, 95% CI: 0.34, 0.96, respectively). For ASD with ID, threatened abortion before 20 weeks gestation and poor fetal growth were associated with an increased risk.

Conclusion

Findings show that indicators of a poor intrauterine environment are associated with an elevated risk of ID, while for ASD, and particularly ASD without ID, the associations are much weaker. As such, these findings highlight the importance of accounting for the absence or presence of ID when examining ASD, if we are to improve our understanding of the causal pathways associated with these conditions.     

A Homogeneous,High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel,Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-³²P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC₅₀ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.