The two Tribolium E(spl) genes show evolutionarily conserved expression and function during embryonic neurogenesis

Tribolium castaneum is a well-characterised model insect, whose short germ-band mode of embryonic development is characteristic of many insect species and differs from the exhaustively studied Drosophila. Mechanisms of early neurogenesis, however, show significant conservation with Drosophila, as a characteristic pattern of neuroblasts arises from neuroectoderm proneural clusters in response to the bHLH activator Ash, a homologue of Achaete–Scute. Here we study the expression and function of two other bHLH proteins, the bHLH-O repressors E(spl)1 and E(spl)3. Their Drosophila homologues are expressed in response to Notch signalling and antagonize the activity of Achaete–Scute proteins, thus restricting the number of nascent neuroblasts. E(spl)1 and 3 are the only E(spl) homologues in Tribolium and both show expression in the cephalic and ventral neuroectoderm during embryonic neurogenesis, as well as a dynamic pattern of expression in other tissues. Their expression starts early, soon after Ash expression and is dependent on both Ash and Notch activities. They act redundantly, since a double E(spl) knockdown (but not single knockdowns) results in neurogenesis defects similar to those caused by Notch loss-of-function. A number of other activities have been evolutionarily conserved, most notably their ability to interact with proneural proteins Scute and Daughterless.     

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d -glycero-d -manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d -glycero-d -manno-heptose-1,7-bisphosphate (αHBP or βHBP) with a strong preference for one anomer (αGmhB or βGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for βHBP but not αHBP, while βGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, βGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from βGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous βGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N.     

Integral strategy for evaluation of fecal indicator performance in bird-influenced saline inland waters

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive Escherichia coli and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that E. coli and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems.     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Affinity chromatography of tryptophan synthase from Escherichia coli. Systematic studies with immobilized tryptophanol phosphate.

Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase. A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme. The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding. The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions. The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands. Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly. Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e. via the active site of alpha subunit. L-Serine is a non-competitive inhibitor of binding. These results are discussed with regard to the composite-active-site hypothesis [T. E. Creighton (1970) Eur. J. Biochem, 13, 1--10]. Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations. Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine. At the low concentrations of the complex found in crude extracts of wild-type E. coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex.     

A model for integrative study of human gastric acid secretion.

We have developed a unique virtual human model of gastric acid secretion and its regulation in which food provides a driving force. Food stimulus triggers neural activity in central and enteric nervous systems and G cells to release gastrin, a critical stimulatory hormone. Gastrin stimulates enterochromaffin-like cells to release histamine, which, together with acetylcholine, stimulates acid secretion from parietal cells. Secretion of somatostatin from antral and corpus D cells comprises a negative-feedback loop. We demonstrate that although acid levels are most sensitive to food and nervous system inputs, somatostatin-associated interactions are also important in governing acidity. The importance of gastrin in acid secretion is greatest at the level of transport between the antral and corpus regions. Our model can be applied to study conditions that are not yet experimentally reproducible. For example, we are able to preferentially deplete antral or corpus somatostatin. Depletion of antral somatostatin exhibits a more significant elevation of acid release than depletion of corpus somatostatin. This increase in acid release is likely due to elevated gastrin levels. Prolonged hypergastrinemia has significant effects in the long term (5 days) by promoting enterochromaffin-like cell overgrowth. Our results may be useful in the design of therapeutic strategies for acid secretory dysfunctions such as hyper- and hypochlorhydria.     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.     

Survival of thermophilic and hyperthermophilic microorganisms after exposure to UV-C,ionizing radiation and desiccation

In this study, we investigated the ability of several (hyper-) thermophilic Archaea and phylogenetically deep-branching thermophilic Bacteria to survive high fluences of monochromatic UV-C (254 nm) and high doses of ionizing radiation, respectively. Nine out of fourteen tested microorganisms showed a surprisingly high tolerance against ionizing radiation, and two species (Aquifex pyrophilus and Ignicoccus hospitalis) were even able to survive 20 kGy. Therefore, these species had a comparable survivability after exposure to ionizing radiation such as Deinococcus radiodurans. In contrast, there was nearly no difference in survival of the tested strains after exposure to UV-C under anoxic conditions. If the cells had been dried in advance of UV-C irradiation, they were more sensitive to UV-C radiation compared with cells irradiated in liquid suspension; this effect could be reversed by the addition of protective material like sulfidic ores before irradiation. By exposure to UV-C, photoproducts were formed in the DNA of irradiated Archaea and Bacteria. The distribution of the main photoproducts was species specific, but the amount of the photoproducts was only partly dependent on the applied fluence. Overall, our results show that tolerance to radiation seems to be a common phenomenon among thermophilic and hyperthermophilic microorganisms.     

A period of transient viremia and occult infection precedes persistent viremia and antiviral immune responses during multiple low-dose intravaginal simian immunodeficiency virus inoculations

In rhesus macaques, classic systemic infection, characterized by persistent viremia and seroconversion, occurred after multiple low-dose (10(3) 50% tissue culture infective doses) intravaginal (IVAG) inoculations with simian immunodeficiency virus (SIV) strain SIVmac251. Monkeys developed classic SIV infections after a variable number of low-dose IVAG exposures to SIVmac251. Once established, the systemic infection was identical to SIV infection following high-dose IVAG SIV inoculation. However, occult systemic infection characterized by transient cell-associated or cell-free viremia consistently occurred early in the series of multiple vaginal SIV exposures. Further, antiviral cellular immune responses were present prior to the establishment of a classic systemic infection in the low-dose vaginal SIV transmission model.