Seed dormancy release and germination characteristics of <i>Corispermum lehmannianum</i> Bunge,an endemic species in the Gurbantunggut desert of China

Seed dormancy release and germination of Corispermum lehmannianum Bunge were tested using various treatments: temperature, cold stratification, gibberelins (GA3), dry storage and sand burial. Results showed that temperature and light did not affect the germination of fresh seeds, cold stratification and GA3 could improve seed germination, whereas dry storage and sand burial did not. The germination percentage was highest at 35/20 °C after the cold stratification and GA3 treatments. Corispermum lehmannianum seeds were classified as non-deep, Type-2, physiological dormancy (PD), whose seed dormancy could be released by cold stratification and GA3.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus adsorption. Virus incubated with soluble alphaVbeta6 had a lower sedimentation rate than native virus on sucrose density gradients, but the particles retained all of their structural proteins and still contained bound integrin and, therefore, were not exhibiting characteristics of a picornavirus A particle.     

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.     

Analysis of neutralizing antigenic sites on the surface of type A12 foot-and-mouth disease virus.

A series of seven neutralizing monoclonal antibodies (nMAbs) directed against type A12 foot-and-mouth disease virus was used to generate neutralization-resistant variants. Both plaque reduction neutralization and microneutralization assays showed that the variants were no longer neutralized by the nMAbs used to generate them, although some of the variants still reacted with the nMAbs at high antibody concentrations. Results of cross-neutralization studies by both plaque reduction neutralization and microneutralization assays suggested the presence of at least one immunodominant antigenic site on the surface of type A12 foot-and-mouth disease virus, along with evidence of a second antigenic site on the viral surface. Two of the variants had reduced virulence in tissue culture as evidenced by their inability to inhibit cellular protein synthesis and a marked reduction in virus-induced cellular morphological alterations. Nucleotide sequencing of the variant genomes placed three epitopes of the major antigenic site on VP1 and the fourth epitope on VP3 and VP1. The one epitope of the minor site appears to reside only on VP1.