Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT)

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC₅₀ values of 0.15?nM against T. congolense and 1.3?nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10?mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.     

Cellular components of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: immunohistochemical (MHC class II) and electron-microscopic observations

This report deals with the distribution, morphology and specific topical relationships of bone-marrow-derived cells (free cells) in the spinal meninges and dorsal root ganglia of the normal rat. The morphology of these cells has been studied by transmission and scanning electron microscopy. Cells expressing the major histocompatibility complex (MHC) class II gene product have been recognized by immunofluorescence. At the level of the transmission electron microscope, free cells are found in all layers of the meninges. Many of them display characteristic ultrastructural features of macrophages, whereas others show a highly vacuolated cytoplasm and are endowed with many processes. These elements lack a conspicuous lysosomal system and might represent dendritic cells. Scanning electron microscopy has revealed that free cells contact the cerebrospinal fluid via abundant cytoplasmic processes that cross the cell layers of the pia mater and of the arachnoid. Cells expressing the MHC class II antigen are also found in all layers of the meninges. They are particularly abundant in the layers immediately adjacent to the subarachnoid space, in the neighbourhood of dural vessels, along the spinal roots and in the dural funnels. In addition to the meninges, strong immunoreactivity for MHC class II antigen is observed in the dorsal root ganglia. The ultrastructural and immunohistochemical findings of this study suggest the existence of a well-developed system of immunological surveillance of the subarachnoid space and of the dorsal root ganglia.     

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.     

A molecular genetic approach to the identification of isochromosomes of chromosome 21

Summary The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.     

Breaking barriers? Ethnicity and socioeconomic background impact on early career progression in the fields of ecology and evolution

The academic disciplines of Science, Technology, Engineering and Mathematics (STEM) have long suffered from a lack of diversity. While in recent years there has been some progress in addressing the underrepresentation of women in STEM subjects, other characteristics that have the potential to impact on equality of opportunity have received less attention. In this study, we surveyed 188 early career scientists (ECRs), defined as within 10 years of completing their PhD, in the fields of ecology, evolutionary biology, behaviour, and related disciplines. We examined associations between ethnicity, age, sexual orientation, sex, socioeconomic background, and disability, with measures of career progression, namely publication record, number of applications made before obtaining a postdoc, type of contract, and number of grant applications made. We also queried respondents on perceived barriers to progression and potential ways of overcoming them. Our key finding was that socioeconomic background and ethnicity were associated with measures of career progression. While there was no difference in the number of reported first‐authored papers on PhD completion, ethnic minority respondents reported fewer other‐authored papers. In addition, ECRs from a lower socioeconomic background were more likely to report being in teaching and research positions, rather than research‐only positions, the latter being perceived as more prestigious by some institutions. We discuss our findings in the context of possible inequality of opportunity. We hope that this study will stimulate wider discussion and help to inform strategies to address the underrepresentation of minority groups in the fields of ecology and evolution, and STEM subjects more widely.     

The Role of PML in the Nervous System

The promyeloctic leukemia protein PML is a tumor suppressor that was originally identified due to its involvement in the (15;17) translocation of acute promyelocytic leukemia. While the majority of early research has focused upon the role of PML in the pathogenesis of leukemia, more recent evidence has identified important roles for PML in tissues outside the hemopoietic system, including the central nervous system (CNS). Here, we review recent literature on the role of PML in the CNS, with particular focus on the processes of neurodevelopment and neurodegeneration, and propose new lines of investigation.     

Spatial segregation of specialists and generalists in bird communities

Each species generally has a close relationship with one or more habitats and can therefore be classified as either specialist or generalist. We studied whether specialist and generalist species are spatially distributed independently of each other. Repeating the analysis for 100 of the most frequent terrestrial bird species recorded over the 10 000 sampled sites of the French Breeding Bird survey, we found that specialists were more abundant if the rest of the community was specialized, and that the inverse was also true. This pattern was far subtler than just a simple dichotomy: most species actually presented a maximum abundance at a value of community specialization similar to their own level of specialization. Bird communities appear very well defined along a specialist–generalist gradient. We believe this pattern becomes more apparent with habitat degradation. The consequences on both ecological services and community resilience may well be considerable.     

Delimiting species: comparing methods for Mendelian characters using lizards of the Sceloporus grammicus (Squamata: Phrynosomatidae) complex

Species form the fundamental units of analysis in many areas of biology and, therefore, rigorous delimitation of this unit is important to a broad array of researchers. Recently, many new empirical methods have been proposed to delimit species in nature, and a large literature exists on the theoretical merit and superiority of each method. However, few empirical studies actually compare the results of these methods applied in the same study system. We used a large allozyme and chromosome dataset to apply a number of genetic-distance, character-based, and tree-based methods to a well-studied, data-rich system: the Sceloporus grammicus lizard complex of central Mexico. We hypothesized species boundaries under a general lineage or evolutionary species conceptual framework in an a priori fashion using mapped restriction-site data (mitochondrial DNA and nuclear rDNA), allozymes, and morphology. We then compared the ability of different methods to recover the "hypothesized evolutionary species" (HES). Highton's genetic-distance method and a tree-based method consistently recovered all four HES, although sometimes with weak support. With two exceptions, other methods recovered the same HES, but additional groups were weakly delimited and nested within the HES. Given the apparent recent divergence of some of the chromosome races and distinct populations in this complex, these are encouraging results. We emphasize the value of specifying testable criteria as clearly as possible and testing these with methods that make use of different properties of a single dataset.