Neuroadapted yellow fever virus strain 17D: a charged locus in domain III of the E protein governs heparin binding activity and neuroinvasiveness in the SCID mouse model

A molecular clone of yellow fever virus (YFV) strain 17D was used to identify critical determinants of mouse neuroinvasiveness previously localized to domain III of the neuroadapted SPYF-MN virus envelope protein. Three candidate virulence substitutions (305F→V, 326K→E, and 380R→T) were individually evaluated for their roles in this phenotype in a SCID mouse model. The virus containing a glutamic acid residue at position 326 of the envelope protein (326E) caused rapidly lethal encephalitis, with a mortality rate and average survival time resembling those of the parental SPYF-MN virus. Determinants at positions 380 (380T) and 305 (305V) did not independently affect neuroinvasiveness. Testing a panel of viruses with various amino acid substitutions at position 326 revealed that attenuation of neuroinvasiveness required a positively charged residue (lysine or arginine) at this position. Molecular-modeling studies suggest that residues 326 and 380 contribute to charge clusters on the lateral surface of domain III that constitute putative heparin binding sites, as confirmed by studies of heparin inhibition of plaque formation. The neuroinvasiveness of YFVs in the SCID model correlated inversely with sensitivity to heparin. These findings establish that residue 326 in domain III of the E protein is a critical determinant of YFV neuroinvasiveness in the SCID mouse model. Together with modeling of domain III from virulent YFV strains, the data suggest that heparin binding activity involving lysine at position 326 may be a modulator of YFV virulence phenotypes.     

Envelope determinants of equine infectious anemia virus vaccine protection and the effects of sequence variation on immune recognition

A highly effective attenuated equine infectious anemia virus (EIAV) vaccine (EIAV(D9)) capable of protecting 100% of horses from disease induced by a homologous Env challenge strain (EIAV(PV)) was recently tested in ponies to determine the level of protection against divergent Env challenge strains (J. K. Craigo, B. S. Zhang, S. Barnes, T. L. Tagmyer, S. J. Cook, C. J. Issel, and R. C. Montelaro, Proc. Natl. Acad. Sci. USA 104:15105-15110, 2007). An inverse correlation between challenge strain Env variation and vaccine protection from disease was observed. Given the striking differences in protective immunity, we hypothesized that analysis of the humoral and cellular immune responses to the Env protein could reveal potential determinants of vaccine protection. Neutralization activity against the homologous Env or challenge strain-specific Env in immune sera from the vaccinated ponies did not correlate with protection from disease. Cellular analysis with Env peptide pools did not reveal an association with vaccine protection from disease. However, when individual vaccine-specific Env peptides were utilized, eight cytotoxic-T-lymphocyte (CTL) peptides were found to associate closely with vaccine protection. One of these peptides also yielded the only lymphoproliferative response associated with protective immunity. The identified peptides spanned both variable and conserved regions of gp90. Amino acid divergence within the principal neutralization domain and the identified peptides profoundly affected immune recognition, as illustrated by the inability to detect cross-reactive neutralizing antibodies and the observation that certain peptide-specific CTL responses were altered. In addition to identifying potential Env determinants of EIAV vaccine efficacy and demonstrating the profound effects of defined Env variation on immune recognition, these data also illustrate the sensitivity offered by individual peptides compared to peptide pools in measuring cellular immune responses in lentiviral vaccine trials.     

Prairie grasses as hosts of the northern corn rootworm (Coleoptera: Chrysomelidae)

We evaluated 27 prairie grass species thought to be among those dominant 200 yr ago in the northern midwest as larval hosts of the northern corn rootworm, Diabrotica barberi Smith and Lawrence. Maize (Zea mays L.), spring wheat (Triticum aestivum L.), and grain sorghum (Sorghum bicolor L.) were included as controls for a total of 30 species. Twenty pots of each species were planted in a randomized complete block design. Each pot was infested 5 wk later with 20 neonate northern corn rootworm larvae. Two pots within each species and block were assigned an extraction date of 7 or 14 d after infestation. The remaining two pots from each block were used to monitor adult emergence. The percentage of larvae recovered, change in larval head capsule width, and change in average dry weights varied significantly among the grass species. The highest percentage of larvae was recovered from slender wheatgrass, Elymus trachycaulus (Link), and this was significantly greater than the percentage recovered from all other species including maize for the 14-d sample date. Several additional species were also relatively good hosts, in that the percentage of larvae recovered from these species was not significantly different from maize. The average dry weight of larvae recovered was significantly greater for larvae recovered from maize than for larvae recovered from all other species except slender wheatgrass, when the two samples dates were combined. Overall, adults were produced from only 6 of the 28 species evaluated, and no analysis was performed because of the low numbers. The results of this study are discussed in relation to the potential of alternate hosts of northern corn rootworm to serve as a bridge to survival on transgenic maize.     

Analysis of cortical arrays from Tradescantia virginiana at high resolution reveals discrete microtubule subpopulations and demonstrates that confocal images of arrays can be misleading

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.     

Identification of coding polymorphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel.

STUDY OBJECTIVE: In this study, the exonic regions of the circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS were re-sequenced and coding changes identified in a panel of 95 individuals varying in ethnicity. STUDY PARTICIPANTS: DNA screening panel consisting of 95 DNA samples (17 American Caucasians, 17 African Americans, 8 Ashkenazi Jews, 8 Chinese, 8 Japanese, 5 Mexican Indians, 8 Mexicans, 8 Northern Europeans, 8 Puerto Ricans, and 8 South Americans) selected from the Coriell Institute Human Variation Panel. RESULTS: In addition to coding changes already identified in the database dbSNP, novel coding changes were identified, including PER1: Pro37Ser, Pro351Ser, Gln988Pro, Ala998Thr; PER2: Leu83Arg, Leu157Leu, Thre174Ile, Phe400Phe, Pro822Pro, Ala828Thr, Ala861Val, Phe876Leu, Val883Met, Val903Ile, Ala923Pro; PER3: Pro67Pro, Val90Ile, His638His, Ala820Ala, Leu929Leu; ARNTL: Arg166Gln, Ser459Phe; CLOCK: Ala34Ala, Ser208Cys, Phe233Phe, Ser632Thr, Ser816Ser; TIMELESS: Met870Val and CRY2: His35His. No coding polymorphisms were identified in CRY1. CONCLUSIONS: Considerable genetic variation occurs within the coding region of the genes regulating circadian rhythm. Many of the non-synonymous coding polymorphisms could affect protein structure/function with the potential to affect molecular regulation of the sleep/wake cycle. Many of the potential functional effects could be ethnic group specific.     

Foot mucus is a good source for non-destructive genetic sampling in Polyplacophora

A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.     

Hydrothermal vents and the origin of life

Submarine hydrothermal vents are geochemically reactive habitats that harbour rich microbial communities. There are striking parallels between the chemistry of the H(2)-CO(2) redox couple that is present in hydrothermal systems and the core energy metabolic reactions of some modern prokaryotic autotrophs. The biochemistry of these autotrophs might, in turn, harbour clues about the kinds of reactions that initiated the chemistry of life. Hydrothermal vents thus unite microbiology and geology to breathe new life into research into one of biology's most important questions - what is the origin of life?