A comparison of alpha and beta diversity patterns of ferns,bryophytes and macrolichens in tropical montane forests of southern Ecuador

We present a first comparison of patterns of alpha and beta diversity of ferns, mosses, liverworts and macrolichens in neotropical montane rainforests, and explore the question whether specific taxa may be used as surrogates for others. In three localities in southern Ecuador, we surveyed terrestrial and epiphytic species assemblages in ridge and slope forests in 28 plots of 400 m2 each. The epiphytic habitat was significantly richer in ferns, liverworts, and macrolichens than the terrestrial habitat; mosses, however, were primarily terrestrial. Alpha diversity of ferns and of liverworts was congruent in both habitats. Mosses were similar to ferns and liverworts only in the epiphytic habitat. Macrolichens did not share patterns of alpha diversity with any other group. Beta diversity of ferns, mosses and liverworts (lichens excluded due to low species richness) was similar in the terrestrial habitat, but not in the epiphytic habitat. Our results demonstrate that patterns of alpha diversity of the studied taxa cannot be used to predict patterns of beta diversity. Moreover, diversity patterns observed in epiphytes are different from terrestrial plants. We noted a general coincidence in species patterns of liverworts and ferns. Diversity patterns of macrolichens, in contrast, were completely independent from any other taxonomic group studied.     

Patterns of extensive genetic differentiation and variation among European harbor seals (Phoca vitulina vitulina) revealed using microsatellite DNA polymorphisms

The harbor seal (Phoca vitulina) has the most extensive distribution of any phocid seal species. An analysis of population structure in this species across its European range was made using 7 phocid derived microsatellites in a sample of 1,029 individuals from 12 separate geographic areas. Despite the species potential for long-distance movement, significant genetic differentiation between areas was observed using an unbiased estimator of RST. Six distinct population units were identified: Ireland-Scotland, English east coast, Waddensea, western Scandinavia (Norway-Kattegat-Skagerrak-west Baltic), east Baltic, and Iceland. Little local substructuring is present along coastlines with a continuous distribution of breeding animals, but differentiation does increase with geographic distance. The degree of differentiation is greater over equivalent distances where the distribution is discontinuous, such as along coasts where breeding colonies are separated by large distances or by stretches of open sea. Patterns of population differentiation derived from microsatellites are very similar to those obtained from previous mitochondrial DNA analysis and suggest that philopatry in harbor seals operates over 300-500 km. In Europe, harbor seals have experienced a complex demographic history and patterns of population structure are likely to have been affected by natural environmental influences such as Pleistocene glaciations and epizootics. Comparison of Nm values from an unbiased estimator of RST, GST, and theta are consistent and, in some cases, may indicate populations where conditions deviate from the expectations of the RST model.      

Role of ascorbate in oxidative protein folding

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.     

Recombinant yellow fever viruses are effective therapeutic vaccines for treatment of murine experimental solid tumors and pulmonary metastases

We have genetically engineered an attenuated yellow fever (YF) virus to carry and express foreign antigenic sequences and evaluated the potential of this type of recombinant virus to serve as a safe and effective tumor vaccine. Live-attenuated YF vaccine is one of the most effective viral vaccines available today. Important advantages include its ability to induce long-lasting immunity, its safety, its affordability, and its documented efficacy. In this study, recombinant live-attenuated (strain 17D) YF viruses were constructed to express a cytotoxic T-lymphocyte epitope derived from chicken ovalbumin (SIINFEKL). These recombinant viruses replicated comparably to the 17D vaccine strain in cell culture and stably expressed the ovalbumin antigen, and infected cells presented the antigen in the context of major histocompatibility complex class I. Inoculation of mice with recombinant YF virus elicited SIINFEKL-specific CD8(+) lymphocytes and induced protective immunity against challenge with lethal doses of malignant melanoma cells expressing ovalbumin. Furthermore, active immunotherapy with recombinant YF viruses induced regression of established solid tumors and pulmonary metastases. Thus, recombinant YF viruses are attractive viral vaccine vector candidates for the development of therapeutic anticancer vaccines.     

Mapping of Functional Elements in the Stem-Anchor Region of Tick-Borne Encephalitis Virus Envelope Protein E

Envelope protein E of the flavivirus tick-borne encephalitis virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino-acid-long protein has been shown to differ significantly from that of other viral fusion proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E proteins were examined. Full-length E proteins and proteins lacking the second of two predicted transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E proteins. Sites located within a predicted alpha-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E proteins. A separate site in the stem, also corresponding to a predicted alpha-helix (amino acids 401 to 413), was essential for the conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus assembly.     

The effect of A23187 on glucose production from methylglyoxal and pyruvate in isolated murine hepatocytes.

1. A23187 increased the glucose production from methylglyoxal in isolated hepatocytes, and maximal stimulation was obtained at 10(-6) M. The effect of A23187 was dependent on the presence of Ca2+. 2. Glucose production from pyruvate (less than 1 mM) in isolated hepatocytes was stimulated by A23187 in the presence of 2.5 mM Ca2+ and was depressed at pyruvate concentrations above 1 mM. Both the virtual Km and the virtual Vmax of glucose production from pyruvate were decreased by A23187.     

Incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line

RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon NdDeltaME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 x 10(7)/ml in the supernatant of these cells. Another replicon (NdDeltaCME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the NdDeltaME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.