The carboxy-terminal sequence of the pestivirus glycoprotein E(rns) represents an unusual type of membrane anchor

The E(rns) protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E(rns) represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E(rns). By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E(rns) is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.     

Scent-marking behaviour in response to conspecific odours by the rat, Rattus norvegicus

The scent-marking behaviour of male and female rats in response to conspecific odours was investigated in two experiments. The results of the first experiment indicated that exposure to conspecific odours generally led to an increase in marking rates. However, no sex differences were found, and large differences in marking responses according to the sex of the odour donor were only found for dioestrous females. These results do not suggest that the primary function of scent-marking in this species is sexual. In the second experiment, odour sources differed not only in the sex of the donor, but also in their relative familiarity to the animal being tested. This experiment showed that males marked more in response to odours from familiar (non-related) females. Females did not differ significantly on measures of marking but did display greater interest in odours from unfamiliar males. The results are discussed in relation to possible functions of marking in this species.     

Effects of estradiol and progesterone on scent-marking behavior of female rats

The effect of estradiol and progesterone on scent-marking behavior of the female rat is reported. Estradiol followed by progesterone injection to ovariectomised rats results in an increase in marking rates. This suggests an endocrine base to the changes in scent-marking behavior that are known to occur with the rat estrous cycle.     

Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis

The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (K_D) of ∼ 0.29  and ∼ 29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle.     

Quantity and safety vs. quality and performance: conflicting interests during mass rearing and transport affect the efficiency of sterile insect technique programs

The sterile insect technique (SIT) requires production of large quantities of sterile males able to successfully compete with wild males for wild females. During eradication of a pest population, the release of fertile insects or capture of non‐marked released flies can have deleterious effects and trigger costly control measures. These perceived risks encourage program managers to apply high radiation doses and high doses of marking dye. In addition, mass rearing factories are strategically located away from release areas to prevent escape of fertile individuals within eradicated areas, raising the need for lengthy transport. Such is the case for Anastrepha obliqua Macquart (Diptera: Tephritidae) released in mango producing areas of Mexico under an SIT‐based eradication campaign. Here, we examined several standard quality‐control parameters for mass‐reared A. obliqua subjected to various time periods under hypoxia during transport, marked with different doses of fluorescent dye, and subjected to different radiation doses. Such factors were evaluated in isolation and in conjunction. Overall, long periods of hypoxia, high marking doses, and high radiation doses reduced the number of flying adults and increased the number of non‐emerged pupae. Some quality‐control parameters such as number of deformed adults, part‐emerged pupae, and non‐flying adults provided less informative guidance or redundant information of fly performance. Some tests such as mortality under stress and mating propensity in small cages were useless in detecting differences in quality among treatments for parameters evaluated during experiments. We discuss the quantity/safety‐quality/performance conflict during eradication using SIT, propose different strategies according to different stages during eradication (management, suppression, eradication, outbreaks in free areas), where males irradiated at low doses and marked with low doses of dye can be released during early suppression, and examine the pertinence of carrying out different quality‐control tests.     

The mouse IAPE endogenous retrovirus can infect cells through any of the five GPI-anchored Ephrin A proteins

The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family -but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.     

Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.     

Immunological Detection of Nitrospira-like Bacteria in Various Soils

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.