Molecular cloning of the cDNA encoding a murine sialic acid-specific 9-O-acetylesterase and RNA expression in cells of hematopoietic and non-hematopoietic origin.

We describe the isolation of a cDNA encoding a murine sialic acid-specific 9-O-acetylesterase as well as its expression pattern in cells of both hematopoietic and non-hematopoietic origin. This enzyme catalyzes the removal of O-acetyl ester groups from position 9 of the parent sialic acid N-acetylneuraminic acid. The cDNA is 2105 nt in length and encodes a protein of 541 amino acids with a predicted molecular weight of 61 kDa, not including oligosaccharides linked to eight potential N-glycosylation sites. The cDNA encoding the acetylesterase displays a widespread distribution in various cell lines and tissues. Expression studies of B lineage cell lines and primary fetal liver cells revealed a developmentally regulated expression pattern in cells of hematopoietic origin. Given the importance of 9-O-acetylation of sialic acids, the cloning of the cDNA encoding a sialic acid-specific 9-O-acetylesterase will be helpful in understanding further the regulation of this post-translational modification and the biological consequences thereof.     

A Rodent Model of Chikungunya Virus Infection in RAG1 -/- Mice,with Features of Persistence,for Vaccine Safety Evaluation

Chikungunya virus (CHIKV) is a positive sense, single stranded RNA virus in the genus Alphavirus, and the etiologic agent of epidemics of severe arthralgia in Africa, Asia, Europe and, most recently, the Americas. CHIKV causes chikungunya fever (CHIK), a syndrome characterized by rash, fever, and debilitating, often chronic arthritis. In recent outbreaks, CHIKV has been recognized to manifest more neurologic signs of illness in the elderly and those with co-morbidities. The syndrome caused by CHIKV is often self-limited; however, many patients develop persistent arthralgia that can last for months or years. These characteristics make CHIKV not only important from a human health standpoint, but also from an economic standpoint. Despite its importance as a reemerging disease, there is no licensed vaccine or specific treatment to prevent CHIK. Many studies have begun to elucidate the pathogenesis of CHIKF and the mechanism of persistent arthralgia, including the role of the adaptive immune response, which is still poorly understood. In addition, the lack of an animal model for chronic infection has limited studies of CHIKV pathogenesis as well as the ability to assess the safety of vaccine candidates currently under development. To address this deficiency, we used recombination activating gene 1 (RAG1^-/-) knockout mice, which are deficient in both T and B lymphocytes, to develop a chronic CHIKV infection model. Here, we describe this model as well as its use in evaluating the safety of a live-attenuated vaccine candidate.     

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.     

A sex-chromosome mutation in Silene latifolia

Silene latifolia is dioecious, yet rare hermaphrodites have been found, and such natural mutants can provide valuable insight into genetic mechanisms. Here, we describe a hermaphrodite-inducing mutation that is almost certainly localized to the gynoecium-suppression region of the Y chromosome in S. latifolia. The mutant Y chromosome was passed through the megaspore, and the presence of two X chromosomes was not necessary for seed development in the parent. This result supports a lack of degeneration of the Y chromosome in S. latifolia, consistent with the relatively recent formation of the sex chromosomes in this species. When crossed to wild-type plants, hermaphrodites performed poorly as females, producing low seed numbers. When hermaphrodites were pollen donors, the sex ratio of offspring they produced through crosses was biased towards females. This suggests that hermaphroditic S. latifolia would fail to thrive and potentially explains the rarity of hermaphrodites in natural populations of S. latifolia. These results indicate that the Y chromosome in Silene latifolia remains very similar to the X, perhaps mostly differing in the primary sex determination regions.     

Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270

Acidithiobacillus ferrooxidans ATCC 23270 possesses two copies of form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The nucleotide sequence identity between the two large and two small subunit peptides was 75% and 58%, respectively. It is proposed that the two copies resulted from lateral gene transfer. Received: 27 October 2000 / Accepted 7 December 2001     

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.     

Endotoxin priming of neutrophils requires endocytosis and NADPH oxidase-dependent endosomal reactive oxygen species

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.     

5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole as a potential PET radioligand for imaging cerebral α7-nAChR in mice

The radiosynthesis and in vivo evaluation of 5-(5-(6-[(11)C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole [(11)C]rac-(1), a potential PET tracer for α7 nicotinic acetylcholine receptors (α7-nAChR), are described. Syntheses of the nonradioactive standard rac-1 and corresponding desmethyl precursor 7 were achieved in several reaction steps. Radiomethylation of 7 with [(11)C]CH(3)I afforded [(11)C]rac-1 in an average radiochemical yield of 30 ± 5% (n=5) with high radiochemical purity and an average specific radioactivity of 444 ± 74 GBq/μmol (n=5). The total synthesis time was 30 min from end-of-bombardment. Biodistribution studies in mice showed that [(11)C]rac-1 penetrates the blood-brain barrier and specifically labels neuronal α7-nAChRs.