Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.     

A claim in search of evidence: reply to Manger’s thermogenesis hypothesis of cetacean brain structure

In a recent publication in Biological Reviews, Manger (2006) made the controversial claim that the large brains of cetaceans evolved to generate heat during oceanic cooling in the Oligocene epoch and not, as is the currently accepted view, as a basis for an increase in cognitive or information‐processing capabilities in response to ecological or social pressures. Manger further argued that dolphins and other cetaceans are considerably less intelligent than generally thought. In this review we challenge Manger’s arguments and provide abundant evidence that modern cetacean brains are large in order to support complex cognitive abilities driven by social and ecological forces.     

Ubiquitin activated tumor necrosis factor receptor associated factor-6 (TRAF6) is recycled via deubiquitination

Ubiquitination of intermediates in the interleukin-1 (IL-1) signaling cascade plays an important role in activation and regulation of the pathway. Both IL-1 receptor associated kinase-1 (IRAK1) and inhibitor of nuclear factor kappaB-alpha (IkappaBalpha) are rapidly ubiquitinated and degraded. Tumor necrosis factor associated factor-6 (TRAF6) is an ubiquitin ligase that is activated by ubiquitination and a signaling intermediate between IRAK1 and IkappaBalpha. It is unknown whether activated TRAF6 is subsequently degraded. We show that in liver cells IL-1 stimulates TRAF6 poly-ubiquitination. In less than 1 h levels of non-modified TRAF6 return to levels near those observed prior to activation. TRAF6 cannot be reactivated in cells which have been pretreated with IL-1. This observation correlates with decreased levels of IRAK1 in IL-1 pretreated cells. The re-establishment of non-modified TRAF6 levels following activation does not require de novo protein synthesis, strongly suggesting that TRAF6 is recycled via deubiquitination. This indicates a unique mechanism of regulation of TRAF6 activity.     

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.     

Geographical patterns of genetic variation in the world collections of wild annual Cicer characterized by amplified fragment length polymorphisms

Cicer reticulatum, C. echinospermum, C. bijugum, C. judaicum, C. pinnatifidum, C. cuneatum and C. yamashitae are wild annual Cicer species and potential donors of valuable traits to improve chickpea (C. arietinum). As part of a large project to characterize and evaluate wild annual Cicer collections held in the world gene banks, AFLP markers were used to study genetic variation in these species. The main aim of this study was to characterize geographical patterns of genetic variation in wild annual Cicer germplasm. Phylogenetic analysis of 146 wild annual Cicer accessions (including two accessions in the perennial C. anatolicum and six cultivars of chickpea) revealed four distinct groups corresponding well to primary, secondary and tertiary gene pools of chickpea. Some possible misidentified or mislabelled accessions were identified, and ILWC 242 is proposed as a hybrid between C. reticulatum and C. echinospermum. The extent of genetic diversity varied considerably and was unbalanced between species with greatest genetic diversity found in C. judaicum. For the first time geographic patterns of genetic variation in C. reticulatum, C. echinospermum, C. bijugum, C. judaicum and C. pinnatifidum were established using AFLP markers. Based on the current collections the maximum genetic diversity of C. reticulatum, C. echinospermum, C. bijugum and C. pinnatifidum was found in southeastern Turkey, while Palestine was the centre of maximum genetic variation for C. judaicum. This information provides a solid basis for the design of future collections and in situ conservation programs for wild annual Cicer.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.