Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.     

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.     

Review of Florida Red Tide and Human Health Effects

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue-one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.     

Purification, modeling, and analysis of botulinum neurotoxin subtype A5 (BoNT/A5) from Clostridium botulinum strain A661222

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences--specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.     

Unlocking the barley genome by chromosomal and comparative genomics

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.     

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.     

Fractionation of cricket testis nuclei on gradients of colloidal silica for study of basic protein changes during spermiogenesis

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

Discovery and SAR of PF-4693627, a potent,selective and orally bioavailable mPGES-1 inhibitor for the potential treatment of inflammation

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure–activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies.