Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Expression of <Emphasis Type="Italic">Ruminococcus albus</Emphasis> Xylanase Gene (<Emphasis Type="Italic">xyn</Emphasis>A) in <Emphasis Type="Italic">Streptococcus bovis</Emphasis> 12-U-1

The objective of this study was to ligate the xylanase gene A (xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [β-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37°C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood. Received: 24 June 2002 / Accepted: 7 October 2002     

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.     

Type II NKT cells stimulate diet-induced obesity by mediating adipose tissue inflammation, steatohepatitis and insulin resistance

The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.     

Amelioration of pneumonia with Streptococcus pneumoniae infection by inoculation with a vaccine against highly pathogenic avian influenza virus in a non-human primate mixed infection model

Background  Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria.
Methods  H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles .
Results  Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae . Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant.
Conclusions  Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.     

Naturally occurring deletional mutation in the C-terminal cytoplasmic tail of CCR5 affects surface trafficking of CCR5

CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(-) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221-222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(-) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4(+) T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(-) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.     

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.