Baculovirus entry into human hepatoma cells

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron microscopy studies revealed that virions were located on the cell surface in pits with an electron-dense coating resembling clathrin. In addition, virus particles were found in larger noncoated plasma membrane invaginations and in intracellular vesicles resembling macropinosomes. In double-labeling experiments, virus particles were detected by confocal microscopy in early endosomes at 30 min and in late endosomes starting at 45 min posttransduction. Viruses were also seen in structures specific for early endosomal as well as late endosomal/lysosomal markers by nanogold preembedding immunoelectron microscopy. No indication of viral entry into recycling endosomes or the Golgi complex was observed by confocal microscopy. In conclusion, these results suggest that AcMNPV enters mammalian cells via clathrin-mediated endocytosis and possibly via macropinocytosis. Thus, the data presented here should enable future design of baculovirus vectors suitable for more specific and enhanced delivery of genetic material into mammalian cells.     

Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member

Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed.     

Surgical transfer of in vivo produced farmed European polecat (Mustela putorius) embryos

Surgical embryo transfer of farmed European polecat (Mustela putorius) was investigated as part of an ex situ preservation project. The long-term objective of the project is to develop effective technology for ex situ conservation of the European mink (Mustela lutreola), which is a highly endangered aboriginal European species. Twenty European polecat females, which served as a model species for the European mink, were humanely killed 4-9 days after first mating and embryos were recovered from oviducts and uteri. Donor-recipient pairs (n = 16) were generated by mating the donors (n = 20) once a day for 2 consecutive days with fertile males and by mating the corresponding recipients (n = 16) on the same days with vasectomized males. An embryo recovery rate of 70% (200 recovered embryos/284 corpora lutea) was achieved from 20 donors. Morulae and blastocysts were recovered between Days 5 and 9 after first mating and were regarded as the best developmental stages for uterine embryo transfer. A total of 172 embryos were transferred surgically under general anaesthesia into the ovarian third of the left uterine horn of 16 recipients with a thin glass capillary. Eleven recipients (69%) produced 72 pups equivalent to an average success rate of 42% (72 pups/172 transferred embryos). The average litter size was 4.5 (range 0-9). These results with this model species, farmed European polecat, demonstrate the potential of embryo transfer as an effective method for the preservation of the endangered European mink (M. lutreola). These species are closely related and have a similar reproductive physiology. However, success of applying embryo transfer in conserving European mink is still dependent on further studies both into its reproductive physiology and developing of improved flushing techniques for anaesthetized donors and the successful transfer of frozen-thawed embryos.     

The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 A resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel nine-coiled left-handed parallel beta-roll. Before the beta-roll, the polypeptide loops from one monomer to the rest, and after the beta-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA beta-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.     

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.     

Structural basis for stem cell factor-KIT signaling and activation of class III receptor tyrosine kinases

Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 A crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a 'wrapping' SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs.     

Recovery of Historically Contaminated Watercourse Polluted by the Chemical Wood Industry: EROD Activity in Fish as Biomarker

Despite outstanding process alterations over decades, pulp- and paper-mill-contaminated sediments and continuing exposure by the effluents may still have effects on biota. In this study, ecotoxicological impacts in the boreal watercourse were analyzed by measuring ethoxyresorufin-O-deethylase (EROD) induction from wild fish populations and from experimentally exposed fish. In order to assess the role of sediment-borne chemicals, juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to the surface sediments of Lake Vatianjärvi and Southern Lake Saimaa, both watercourses impacted by the chemical wood industry for approximately a century. Hepatic EROD activity was also measured from roach (Rutilus rutilus) and perch (Perca fluviatilis) caught in Lake Vatianjärvi. Increased EROD activity was not observed in wild fish caught in Lake Vatianjärvi nor in rainbow trout exposed to the sediment of Lake Vatianjärvi, but it was observed in rainbow trout exposed to the sediment of Southern Lake Saimaa, probably explained by critically high concentrations of retene. The results of both the laboratory and field study indicate the absence of EROD-inducing compounds of the previously heavily contaminated Lake Vatianjärvi.     

Seasonal survival of young and adult black grouse females in boreal forests

Direct demographical information from different populations of species in concern may reveal processes to target by management. We estimated seasonal survival rates using data from 253 individual females in a continuous population of black grouse Tetrao tetrix in Central Finland in 2005–2007. The information theoretic model selection included the effects of age class, ecological season, year, and their relevant interactions. We showed a clear difference in the survival of young and older females during late winter, a decrease in female survival during spring, and variation between the study years. The most frequent proximate mortality cause was avian predation, and the avian predation rate was highest in spring.