Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice,but provide minimal protection

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (K(b-/-)D(b-/-) mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (beta(2)m(-/-) mice). The susceptibility of K(b-/-)D(b-/-) mice cannot be explained by the failure of CD8(+) T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8(+) T cells were a relatively small population in uninfected K(b-/-)D(b-/-) mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-gamma following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of K(b-/-)D(b-/-) mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8alpha(-/-) mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8(+) effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.     

Local blockade of allergic airway hyperreactivity and inflammation by the poxvirus-derived pan-CC-chemokine inhibitor vCCI

Allergen-induced asthma is characterized by chronic pulmonary inflammation, reversible bronchoconstriction, and airway hyperreactivity to provocative stimuli. Multiple CC-chemokines, which are produced by pulmonary tissue in response to local allergen challenge of asthmatic patients or experimentally sensitized rodents, chemoattract leukocytes from the circulation into the lung parenchyma and airway, and may also modify nonchemotactic function. To determine the therapeutic potential of local intrapulmonary CC-chemokine blockade to modify asthma, a recombinant poxvirus-derived viral CC-chemokine inhibitor protein (vCCI), which binds with high affinity to rodent and human CC-chemokines in vitro and neutralizes their biological activity, was administered by the intranasal route. Administration of vCCI to the respiratory tract resulted in dramatically improved pulmonary physiological function and decreased inflammation of the airway and the lung parenchyma. In contrast, vCCI had no significant effect on the circulating levels of total or allergen-specific IgE, allergen-specific cytokine production by peripheral lymph node T cells, or peritoneal inflammation after local allergen challenge, indicating that vCCI did not alter systemic Ag-specific immunity or chemoattraction at extrapulmonary sites. Together, these findings emphasize the importance of intrapulmonary CC-chemokines in the pathogenesis of asthma, and the therapeutic potential of generic and local CC-chemokine blockade for this and other chronic diseases in which CC-chemokines are locally produced.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.     

Choriodecidual group B streptococcal inoculation induces fetal lung injury without intra-amniotic infection and preterm labor in Macaca nemestrina

Background

Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero.

Methodology/Principal Findings

Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (n = 5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1×10⁶ colony forming units (n = 5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intra-alveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology.

Conclusions/Significance

A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome.