Calmodulin regulation of Nav1.4 current: role of binding to the carboxyl terminus

Calmodulin (CaM) regulates steady-state inactivation of sodium currents (Na(V)1.4) in skeletal muscle. Defects in Na current inactivation are associated with pathological muscle conditions such as myotonia and paralysis. The mechanisms of CaM modulation of expression and function of the Na channel are incompletely understood. A physical association between CaM and the intact C terminus of Na(V)1.4 has not previously been demonstrated. FRET reveals channel conformation-independent association of CaM with the C terminus of Na(V)1.4 (CT-Na(V)1.4) in mammalian cells. Mutation of the Na(V)1.4 CaM-binding IQ motif (Na(V)1.4(IQ/AA)) reduces cell surface expression of Na(V)1.4 channels and eliminates CaM modulation of gating. Truncations of the CT that include the IQ region abolish Na current. Na(V)1.4 channels with one CaM fused to the CT by variable length glycine linkers exhibit CaM modulation of gating only with linker lengths that allowed CaM to reach IQ region. Thus one CaM is sufficient to modulate Na current, and CaM acts as an ancillary subunit of Na(V)1.4 channels that binds to the CT in a conformation-independent fashion, modulating the voltage dependence of inactivation and facilitating trafficking to the surface membrane.     

Identification of quantitative trait loci affecting cattle temperament

In addition to its potential contribution to improving animal welfare, the study of the genetics of cattle behavior may provide more general insights into the genetic control of such complex traits. We carried out a genome scan in a Holstein x Charolais cross cattle population to identify quantitative trait loci (QTL) influencing temperament-related traits. Individuals belonging to the second-generation of this population (F(2) and backcross individuals) were subjected to 2 behavioral tests. The flight from feeder (FF) test measured the distance at which the animal moved away from an approaching human observer, whereas the social separation (SS) test categorized different activities which the animal engaged in when removed from its penmates. The entire population was genotyped with 165 microsatellite markers. A regression interval mapping analysis identified 29 regions exceeding the 5% chromosome-wide significance level, which individually explained a relatively small fraction of the phenotypic variance of the traits (from 3.8% to 8.4%). One of the significant associations influencing an FF test trait on chromosome 29 reached the 5% genome-wide significance level. Eight other QTL, all associated with an SS test trait, reached the 1% chromosome-wide significance level. The location of some QTL coincided with other previously reported temperament QTL in cattle, whereas those that are reported for the first time here may represent general loci controlling temperament differences between cattle breeds. No overlapping QTL were identified for the traits measured by the 2 different tests, supporting the hypothesis that different genetic factors influence behavioral responses to different situations.     

SAP enables T cells to help B cells by a mechanism distinct from Th cell programming or CD40 ligand regulation

Genetic mutations disrupting the function of signaling lymphocytic activation molecule-associated protein (SAP) lead to T cell intrinsic defects in T cell-dependent Ab responses. To better understand how SAP enables Th cells to help B cells, we first assessed whether molecules important for B cell help are dysregulated in SAP-deficient (SAP knockout (KO)) mice. CD40 ligand (CD40L) expression was enhanced on unpolarized SAP KO T cells; however, Th2 polarization returned their CD40L expression to wild-type levels without rescuing their ability to help B cells. CD40L also localized normally to the site of contact between SAP KO T cells and Ag-bearing B cells. Finally, CD40L-deficient Th cells and SAP KO Th cells differed in their abilities to help B cells in vitro. These data argue that Ab defects caused by SAP deficiency do not result from a loss of CD40L regulation or CD40L function on CD4 T cells. SAP KO Th cells additionally displayed normal patterns of migration and expression of ICOS and CXCR5. Global gene expression was remarkably similar in activated SAP KO vs wild-type T cells, prompting us to investigate whether SAP is necessary for "programming" T cells to become B cell helpers. By restricting SAP expression during differentiation, we determined that SAP is not required during the first 5 days of T cell activation/differentiation to generate Th cells capable of helping B cells. Instead, SAP is necessary for very late stages of differentiation or, most likely, for allowing Th cells to communicate during cognate T:B interactions.     

Liver X receptor agonists increase airway reactivity in a model of asthma via increasing airway smooth muscle growth

The liver X receptors (LXRalpha/beta) are orphan nuclear receptors that are expressed in a large number of cell types and have been shown to have anti-inflammatory properties. Nuclear receptors have previously proved to be amenable targets for small molecular mass pharmacological agents in asthma, and so the effect of an LXR ligand was assessed in models of allergic airway inflammation. LXR agonist, GW 3965, was profiled in rat and mouse models of allergic asthma. In the Brown Norway rats, GW 3965 (3-30 mg/kg) was unable to reduce the bronchoalveolar lavage eosinophilia associated with this model and had no impact on inflammatory biomarkers (eotaxin and IL-1beta). The compound did significantly stimulate ABCA-1 (ATP-binding cassette A1) mRNA expression, indicating that there was adequate exposure/LXR activation. In the mouse model, the LXR ligand surprisingly increased airway reactivity, an effect that was apparent in both the Ag and nonchallenged groups. This increase was not associated with a change in lung tissue inflammation or number of mucus-containing cells. There was, however, a marked increase in airway smooth muscle thickness in both treated groups. We demonstrated an increase in contractile response to exogenous methacholine in isolated airways taken from LXR agonist-treated animals compared with the relevant control tissue. We corroborated these findings in a human system by demonstrating increased proliferation of cultured airway smooth muscle. This phenomenon, if evidenced in man, would indicate that LXR ligands may directly increase airway reactivity, which could be detrimental, especially in patients with existing respiratory disease and with already compromised lung function.     

Danger signaling through the inflammasome acts as a master switch between tolerance and sensitization

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.     

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.