Identification and expression of two novel cytochrome P450 genes, belonging to CYP4 and a new CYP331 family, in the polychaete Capitella capitata sp.I

The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily.     

Newcastle Disease Virus-Vectored Vaccines Expressing the Hemagglutinin or Neuraminidase Protein of H5N1 Highly Pathogenic Avian Influenza Virus Protect against Virus Challenge in Monkeys

H5N1 highly pathogenic avian influenza virus (HPAIV) causes periodic outbreaks in humans, resulting in severe infections with a high (60%) incidence of mortality. The circulating strains have low human-to-human transmissibility; however, widespread concerns exist that enhanced transmission due to mutations could lead to a global pandemic. We previously engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to express the HPAIV hemagglutinin (HA) protein, and we showed that this vaccine (NDV/HA) induced a high level of HPAIV-specific mucosal and serum antibodies in primates when administered through the respiratory tract. Here we developed additional NDV-vectored vaccines expressing either HPAIV HA in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain of influenza virus [HA(RV)], in order to address concerns of enhanced vector replication or genetic exchange, or HPAIV neuraminidase (NA). The three vaccine viruses [NDV/HA, NDV/HA(RV), and NDV/NA] were administered separately to groups of African green monkeys by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 × 10⁷ PFU of homologous HPAIV. Thus, needle-free, highly attenuated NDV-vectored vaccines expressing either HPAIV HA, HA(RV), or NA have been developed and demonstrated to be individually immunogenic and protective in a primate model of HPAIV infection. The finding that HA(RV) was protective indicates that it would be preferred for inclusion in a vaccine. The study also identified NA as an independent protective HPAIV antigen in primates. Furthermore, we demonstrated the feasibility of aerosol delivery of NDV-vectored vaccines.H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in human infections in 1997; previously, it had been found only in birds (11, 50). To date, this virus has been identified in 436 confirmed cases of human infection in 15 countries, 262 (60%) of which were fatal (75). The currently circulating H5N1 strains are characterized by low human-to-human transmissibility. This has been attributed, in part, to a preference for binding to α-2,3-linked sialic acids that are present in high concentrations throughout the avian respiratory tract but were thought to be found primarily in the lower human respiratory tract (57), although this explanation has been questioned (48, 49). It has also been observed that mutations in the PB2 subunit of the viral polymerase are necessary to confer the ability for the virus to be spread by aerosolized nasal droplets in ferrets (72). Whatever factors may be involved, there is widespread concern that the avian virus could mutate to enhance its transmissibility among humans, possibly resulting in a global pandemic (28, 50). For the avian H9N2 virus, which also has pandemic potential, it has been demonstrated that only five amino acid changes were sufficient for the virus to gain the ability to be spread by aerosolized nasal droplets in a ferret model (60). Thus, there is an urgent need for vaccines against HPAIV.Several vaccine strategies for HPAIV have been evaluated (reviewed in references 32 and 41), including inactivated and live attenuated vaccines. These efforts have been hampered by several factors. HPAIV strains are highly virulent for embryonated chicken eggs, the most widely used substrate for vaccine manufacture, and their rapid death following inoculation renders eggs unsuitable for efficient virus propagation. In addition, the major protective antigen, hemagglutinin (HA), administered either as a purified protein or in inactivated HPAIV virions, appears to be poorly immunogenic (69, 70). An additional factor complicating the development of HPAIV vaccines based on inactivated virus is the high cost and biohazard associated with HPAIV propagation, which must be done under enhanced biosafety level 3 (BSL-3) containment, although this problem might be addressed by the use of live attenuated reassortant influenza virus vaccines that contain the HPAIV glycoproteins on the background of an avirulent human influenza virus strain (24, 37). In addition, such reassortant strains might serve directly as live attenuated vaccines. Unfortunately, the latter approach may be limited by subtle and unpredictable incompatibility between the avian-origin glycoproteins and human-origin vaccine backgrounds acceptable for human use, which can result in overattenuation in vivo (24). There are also lingering concerns about the significant potential, with a live HPAIV vaccine, for reassortment between gene segments of the vaccine virus and circulating influenza virus strains, which might result in novel strains with unpredictable biological properties (63).We and others have been evaluating Newcastle disease virus (NDV) as a general human vaccine vector for emerging pathogens, including H5N1 HPAIV (7, 18-20, 29). NDV is an avian paramyxovirus that is antigenically unrelated to common human pathogens; hence, its use in humans should not be affected by host immunity to common pathogens. The many naturally occurring strains of NDV can be categorized into three pathotypes based on virulence in chickens: velogenic strains, causing severe disease with high mortality; mesogenic strains, causing disease of intermediate severity with low mortality; and lentogenic strains, causing mild or inapparent infections (reviewed in reference 2). Lentogenic, and sometimes mesogenic, strains of NDV are in wide use as live attenuated vaccines against velogenic NDV in poultry (2). When mesogenic or lentogenic NDV was administered to the respiratory tracts of nonhuman primates as a model for the immunization of humans, the virus was highly attenuated for replication, was shed only at low titers, appeared to remain restricted to the respiratory tract, and was highly immunogenic for the expressed foreign antigen (7). We recently demonstrated that a mesogenic strain of NDV expressing the HA protein of H5N1 HPAIV (NDV/HA) elicited high titers of neutralizing antibodies in serum following combined intranasal (i.n.) and intratracheal (i.t.) delivery in a nonhuman primate model (20). Vaccination of mice with a similar NDV-vectored vaccine protected them from HPAIV challenge (29). However, results obtained with mice do not reliably predict the efficacy of an influenza virus vaccine for human use, due to the pathophysiological and phylogenetic differences between mice and humans (71). In particular, mice may produce a potent immune response to HPAIV vaccines (64) that may not be reproduced in clinical trials (38). These considerations are especially important for a vaccine based on a live viral vector platform, since its immunogenicity, and therefore its protective efficacy, is directly linked to replication, which can differ greatly in various experimental animals versus humans (reviewed in references 6 and 9). Therefore, the protective efficacy of NDV-based vaccines against HPAIV challenge in nonhuman primate models—the closest model to humans—has remained unknown.The protease recognition sequence of the HA protein is one of the major determinants of avian influenza virus pathogenicity (62). HPAIV strains have a “polybasic” cleavage site, containing multiple basic amino acids, that is readily cleaved by ubiquitous intracellular subtilisin-like proteases, facilitating the replication and spread of the virus. In contrast, the HA cleavage site of low-pathogenicity strains contains fewer basic amino acids and depends on secretory trypsin-like proteases found in the respiratory and enteric tracts, resulting in more-localized infections (30, 62). The presence of a polybasic cleavage site in the H5 HA of any live vaccine raises some concern about the possibility of genetic exchange with circulating strains of influenza virus. It should be noted that genetic exchange involving paramyxoviruses is a rare event (14) that has been documented only once (61). However, elimination of the polybasic HA cleavage site would mitigate the effects of even this rare possibility of genetic exchange. Another concern was based on our previous finding that the HPAIV H5 HA protein is incorporated into the NDV envelope as a trimer (20), consistent with its presence in a functional form. While we previously showed that this did not enhance the pathogenicity of the NDV/HA recombinant in chickens (20), we could not rule out the possibility that it might confer an altered tropism on the NDV/HA virus in other systems. For example, a recombinant parainfluenza virus type 3 expressing the Ebola virus glycoprotein incorporated the foreign protein into its envelope, allowing cellular attachment and fusion of the vaccine virus independently of the vector''s own envelope glycoproteins (10).In addition to the HA protein, the neuraminidase (NA) protein is also present on the surfaces of influenza virus-infected cells and virions. Antibodies specific for NA are not thought to interfere with the initial viral attachment and penetration of host cells (36, 40, 54). However, NA-specific antibodies prevent the release of virus from infected cells, thereby decreasing viral spread (35), and they increase resistance to viral infection in humans (40, 47, 54). They also provide at least some protection against viruses bearing homologous or heterologous NA proteins of the same subtype in a mouse model (12, 56). NA also appears to evolve at a lower rate than HA, suggesting that NA-specific antibodies may provide broader protection than a vaccine utilizing HA alone (39). Therefore, it was important to assess the immunogenicity and protective efficacy of the HPAIV NA independently of those of HA, which has not previously been done in a human or nonhuman primate model.     

IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.     

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.     

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables

A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.     

The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels?

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump gamma-subunit. Originally it was suggested that CHIF could induce the expression of the I(Ks) current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.     

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.