Photosynthetic microbial fuel cells with positive light response

The current study introduces an aerobic single‐chamber photosynthetic microbial fuel cell (PMFC). Evaluation of PMFC performance using naturally growing fresh‐water photosynthetic biofilm revealed a weak positive light response, that is, an increase in cell voltage upon illumination. When the PMFC anodes were coated with electrically conductive polymers, the rate of voltage increased and the amplitude of the light response improved significantly. The rapid immediate positive response to light was consistent with a mechanism postulating that the photosynthetic electron‐transfer chain is the source of the electrons harvested on the anode surface. This mechanism is fundamentally different from the one exploited in previously designed anaerobic microbial fuel cells (MFCs), sediment MFCs, or anaerobic PMFCs, where the electrons are derived from the respiratory electron‐transfer chain. The power densities produced in PMFCs were substantially lower than those that are currently reported for conventional MFC (0.95 mW/m² for polyaniline‐coated and 1.3 mW/m² for polypyrrole‐coated anodes). However, the PMFC did not depend on an organic substrate as an energy source and was powered only by light energy. Its operation was CO₂‐neutral and did not require buffers or exogenous electron transfer shuttles. Biotechnol. Bioeng. 2009; 104: 939–946. © 2009 Wiley Periodicals, Inc.     

Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein

We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner.The transmission of highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype to humans since the year 1997 has caused a high mortality rate of almost 60% (62). Patients infected with H5N1 influenza virus developed mainly severe respiratory disease, characterized by fever, cough, shortness of breath, and pneumonia, that frequently progressed to acute respiratory distress syndrome (ARDS) and multiorgan failure (28, 68, 69). In fatal cases, the median time from onset to death was 9 to 10 days (1). Systemic spread (18) and hypercytokinemia (11) have been described as possible disease-aggravating factors of HPAIV-H5N1 viruses, but the reasons for their high virulence in humans are incompletely understood.Due to the potential pandemic threat presented by H5N1 viruses, there is great interest in the identification of viral virulence determinants and their mode of action. This is critical not only for a better understanding of the pathogenic mechanisms induced by these viruses but also for the development of new drugs to treat the infections. The high virulence of HPAIV-H5N1 isolates in the avian host correlates with the presence of a polybasic cleavage site in the hemagglutinin (HA), facilitating its intracellular cleavage by furin-like proteases (27, 50). Further, amino acid substitutions in the PA protein (T515A) (30) and in the NS1 protein (V149A) (40) have been reported to regulate the virulence of corresponding HPAIV-H5N1 isolates in ducks and chickens. The known molecular determinants of virulence in mammalian hosts also include the polybasic cleavage site in the HA (23) and several polymorphisms in the PB2 polymerase subunit and the proapoptotic PB1-F2 protein. Thus, a serine residue at position 66 in the PB1-F2 protein increased viral replication and decreased survival in the mouse model (9). Also, specific amino acid polymorphisms within PB2 (E627K or D701N) can increase virulence in mice (23, 39) and viral replication in mammalian cells (7, 57, 58). Furthermore, the nonstructural NS1 protein, which has a major function in the inhibition of type I interferon (IFN) (17, 19) and in the limitation of the antiviral effects of IFN-induced proteins, including PKR (4, 22), OAS/RNase L (45), and RIG-I (16, 48, 63, 64), contributes to virulence in mammals (34, 55).The domain structure of the NS1 protein is well characterized; it includes an N-terminal RNA binding and dimerization domain and a nuclear localization signal (NLS) at positions 34 to 38 (summarized in reference 19). The NS1 proteins of most human strains circulating between 1950 and 1986 also contain a second NLS at positions 219 to 227 (NLS-2), which includes four conserved basic amino acids (K219, R220, R224, R227) (44). A large-scale sequence analysis showed that the NS1 proteins of avian and human influenza viruses differ in their C-terminal sequences, indicating possible differences in the associated activity (46). Among most high- and low-pathogenicity avian influenza viruses, the last four NS1 amino acids consist of the conserved sequence ESEV (3,007 of 3,692 isolates described in the NCBI database [3]), while for the majority of seasonal human influenza viruses, the motif RSKV is typical (1,911 of 2,713 isolates). Significantly, only the NS1 protein carrying the “avian” ESEV motif interacted in vitro with 24 cellular factors carrying a PDZ (postsynaptic density protein 95, Drosophila disc large tumor suppressor, and zonula occludens 1 protein) domain. The human genome encodes at least 214 proteins containing one or more of these protein interaction modules that recognize short peptide motifs, which are most often present at the C termini of their targets (36, 38). Many PDZ domain proteins have been shown to mediate the formation and localization of higher-order complexes and to participate in various cellular signaling events regulating, for instance, cell polarity and neuronal function (31). Therefore, it was hypothesized that the abundant expression of “avian” NS1 protein capable of interacting with human PDZ domains could possibly disturb their function and aggravate disease severity in H5N1 infections (46). However, there is only limited experimental support for the universal validity of this hypothesis. The grafting of the “avian” ESEV sequence into the C terminus of NS1 protein expressed by mouse-adapted influenza A/WSN/33 virus (H1N1) decreased the mean lethal dose by about 1 order of magnitude (32). Still, it is not clear to what extent this motif contributes to the virulence of HPAIV-H5N1 and other natural influenza A viruses in avian and mammalian hosts.The goal of the present study was to elucidate the role of the C-terminal NS1 motif in viral replication and disease caused by the prototypic influenza A/Vietnam/1203/04 (VN/1203) virus, isolated in a fatal human case (60). This virus expresses an NS1 protein that is very similar or identical at positions 1 to 215 to homologues expressed by other HPAIV-H5N1 strains but naturally lacks the 10 C-terminal amino acids (aa), including the terminal ESEV motif, due to a premature stop codon (Fig. ​(Fig.1).1). We used reverse genetics to produce a recombinant VN/1203 wild-type (WT) virus and two variants with reconstituted NS1 C termini ending either with the “avian” ESEV or with the “human” RSKV sequence. Experimental infections of mice and chickens revealed that all three viruses caused highly lethal infections in both species, with only moderate differences in viral titers in the organs of the mice. Thus, we show that the C-terminal ESEV motif of the NS1 protein contributes little to the virulence of H5N1 viruses in mice and chickens, and we suggest that this motif modulates viral virulence in a strain- and host-dependent manner.Open in a separate windowFIG. 1.Growth kinetics of recombinant VN/1203 viruses expressing WT or elongated NS1 proteins in human, murine, and avian cells. (A) Scheme of the viral VN/1203-NS1 protein with the RNA binding domain and the nuclear localization signals (NLS) at positions 34 to 38 and 214 to 225 indicated. Amino acids involved in NLS2 function are underlined. The C-terminal sequences of the WT and elongated mutant NS1 proteins are given, and the PL motif is shown in boldface. (B to E) Human A549 alveolar cells, murine NIH 3T3 fibroblasts, chicken embryo fibroblasts (CEFs), or EFB-R1 duck embryo fibroblasts (DEFs) were infected with recombinant VN/1203-WT, -ESEV, or -RSKV viruses at an MOI of 0.001. Aliquots of supernatants were harvested at the indicated time points, and samples were titrated by plaque assays in MDCK cells. (F) Human A549 cells were infected at an MOI of 2, and virus titers in supernatants taken at the indicated time points were determined by plaque assays. Results are averages for at least two independent experiments with biological duplicates. Error bars indicate standard deviations.     

Relevance of histone acetylation and replication timing for deposition of centromeric histone CENP-A

A centromere-specific variant of histone H3, centromere protein A (CENP-A), is a critical determinant of centromeric chromatin, and its location on the chromosome may determine centromere identity. To search for factors that direct CENP-A deposition at a specific chromosomal locus, we took advantage of the observation that CENP-A, when expressed at elevated levels, can get incorporated at ectopic sites on the chromosome, in addition to the centromere. As core histone hypoacetylation and DNA replication timing have been implicated as epigenetic factors that may be important for centromere identity, we hypothesized that the sites of preferential CENP-A deposition will be distinguished by these parameters. We found that, on human dicentric chromosomes, ectopically expressed CENP-A preferentially incorporates at the active centromere only, despite the fact that the levels of histone acetylation and replication timing were indistinguishable at the two centromeres. In CHO cells, ectopically expressed CENP-A is preferentially targeted to some, but not all telomeric regions. Again, these regions could not be distinguished from other telomeres by their acetylation levels or replication timing. Thus histone acetylation and replication timing are not sufficient for specifying the sites of CENP-A deposition and likely for centromere identity.     

Isonymy and the genetic structure of Albanian populations

It is well known that in systems of surname transmission through the paternal line, surnames simulate neutral gene alleles belonging to the Y chromosome. This property of surnames was used to analyze the genetic structure of Albanian populations. Two large samples of surnames belonging to two different periods of time were analyzed. The analysis of indicators of population structure showed that geographical distance has an important effect on surname distribution. It seems that isolation by distance and genetic drift have been still important factors in the determination of the genetic structure of the Albanian population.     

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.     

Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1(-/-) mice, which lacked the pathway's ubiquitin ligase E3alpha, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2(-/-) mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2(-/-) genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2(-/-) females died as embryos, whereas UBR2(-/-) males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2(-/-) testes was normal and spermatogonia were intact as well, but UBR2(-/-) spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2(-/-) spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.     

The absence of Arabidopsis-type telomeres in Cestrum and closely related genera Vestia and Sessea (Solanaceae): first evidence from eudicots

Using slot-blot and fluorescent in situ hybridization (FISH), we found no evidence for the presence of the Arabidopsis-type telomeric sequence (TTTAGGG)n at the chromosome termini in any of the Cestrum species we investigated. Probing for the human-type telomere (TTAGGG)n also revealed no signal. However, polymerase chain reaction experiments indicated that there are short lengths of the sequence TTTAGGG dispersed in the genome but that these sequences are almost certainly too short to act as functional telomeres even if they were at the chromosome termini. An analysis of related genera Vestia and Sessea indicates that they too lack the Arabidopsis-type telomere, and the sequences were lost in the common ancestor of these genera. We found that the Cestrum species investigated had particularly large mean chromosome sizes. We discuss whether this is a consequence of alternative telomere end maintenance systems.     

Nuclear DNA C-values in 30 species double the familial representation in pteridophytes

Nuclear DNA C-values and genome size are important biodiversity characters with fundamental biological significance. Yet C-value data for pteridophytes, a diverse group of vascular plants with approx. 9000 extant species, remain scarce. A recent survey by Bennett and Leitch (2001, Annals of Botany 87: 335-345) found that C-values were reported for only 48 pteridophyte species. To improve phylogenetic representation in this group and to check previously reported estimates, C-values for 30 taxa in 17 families were measured using flow cytometry for all but one species. This technique proved generally applicable, but the ease with which C-value data were generated varied greatly between materials. Comparing the new data with those previously published revealed several large discrepancies. After discounting doubtful data, C-values for 62 pteridophyte species remained acceptable for analysis. The present work has increased the number of such species' C-values by 93 %, and more than doubled the number of families represented (from 10 to 21). Analysis shows that pteridophyte C-values vary approx. 450-fold, from 0-16 pg in Selaginella kraussiana to 72.7 pg in Psilotum nudum var. gasa. Superimposing C-value data onto a robust phylogeny of pteridophytes suggests some possible trends in C-value evolution and highlights areas for future work.     

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.