A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

Homeotic Transformation of Ovules into Carpel-like Structures in Arabidopsis

Ovules are specialized reproductive organs that develop within the carpels of higher plants. In Arabidopsis, mutations in two genes, BELL1 (BEL1) and APETALA2 (AP2), disrupt ovule development. In Bel1 ovules, the inner integument fails to form, the outer integument develops abnormally, and the embryo sac arrests at a late stage of megagametogenesis. During later stages of ovule development, cells of the outer integument of a Bel1 ovule sometimes develop into a carpel-like structure with stigmatic papillae and second-order ovules. The frequency of carpel-like structures was highest when plants were grown under conditions that normally induced flowering and was correlated with ectopic expression in the ovule of AGAMOUS (AG), an organ-identity gene required for carpel formation. Together, these results suggested that BEL1 negatively regulates AG late in ovule development. Likewise, mutants homozygous for the strong AP2 allele ap2-6 sometimes displayed structures with carpel-like features in place of ovules. However, such abnormal Ap2 ovules are much less ovulelike in morphology and form earlier than the Bel1 carpel-like structures. Because one role of the AP2 gene is to negatively regulate AG expression early in flower development, it is possible that AP2 works in a similar manner in the ovule. A novel ovule phenotype observed in Bel1/Ap2-6 double mutants suggested that BEL1 and AP2 genes function independently during ovule development.     

Regeneration of plantlets from embryogenic suspension cultures of pickling cucumber (Cucumis Sativus L. CV. Endeavor)

Summary Procedures have been developed for the initiation and long-term maintenance of embryogenic suspension cultures of pickling cucumber (Cucumis sativus) cultivar Endeavor and for the regeneration of normal plantlets. Embryogenic calluses from petiole explants plated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), both at 5μM, were used to initiate the embryogenic suspension cultures. Among various growth regulator combinations evaluated for initiation and maintenance of these suspension cultures, only MS medium with 2,4-D and BA, both at 1μM, produced cultures that were yellow, friable, and still regenerable after repeated subculture (every two wk) over a 3- to 15-mo. period. The effects of various concentrations of auxin and cytokinin in the plating medium, the addition of AgNO₃, and various plating procedures were also evaluated. The highest frequency of regeneration of shoots and plantlets was achieved by plating aggregates onto filter paper overlaid on MS medium with naphthalene acetic acid (NAA)/BA at a concentration of 2:1 or 1:1μM. The addition of activated charcoal (0.5%) or AgNO₃ (30μM) in the plating medium did not enhance the frequency of plantlet regeneration. The highest frequency of normal-appearing plantlets recovered was 42 to 46% per petri dish. The procedures described in this study can be used to increase plantlet recovery from individual embryogenic calluses of pickling cucumber.     

Studies on atrophic change of soleus muscle and its countermeasures in suspended rat

In the environment of microgravity, the disused atrophy of skeletal muscle, especially leg's muscle, would occur. The three purposes of this study were: 1. To observe the dynamic changes of disused atrophy of skeletal muscle under simulated weightlessness; 2. To approach the mechanism of disused atrophy of muscle; 3. To approach the countermeasures for reducing the degree of atrophy of muscle.     

The effect of submergence on tuber production and dormancy in two subspecies ofBolboschoenus maritimus

Two subspecies ofBolboschoenus maritimus (L.)Palla were cultivated in submerged and non-submerged conditions. The production of tubers, the proportion of tubers remaining dormant and the time between production and reactivation were investigated. Total tuber production was found to be dependent on subspecies and may be considered a biological character of the subspecies.Bolboschoenus maritimus ssp.compactus, a subspecies of less, flooded habitats, produced more tubers with a greater capacity for growth leaving a smaller proportion of dormant tubers. Inactivity of tubers and their reactivation after one or two years were enhanced by submergence in both subspecies. The proportion of tubers that are permanently dormant as well as the dormancy of the whole plant are important features influencing the survival of populations ofB. maritimus over periods of unfavourable conditions.     

Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.