An Epidermal Growth Factor-like Repeat of Del1 Protein Increases the Efficiency of Gene Transfer In Vitro

In this paper, we found that Del1, an extracellular matrix protein secreted by embryonic endothelial cells, increases the efficiency of transfection in vitro. Conditioned medium containing Del1 increased transfection by the LacZ gene using several non-viral gene transfer systems, including lipoplex and polyplex methods. Experiments using deletion mutants and fragments of Del1 revealed that the third epidermal growth factor-like repeat (E3) of Del1 mediates the enhancement of gene transfer and, furthermore, that the motif CXDXXXFXCXC is essential. Incubation of Pro5 cells, a yolk sac-derived cell line, with as low as 16 pM recombinant E3 was sufficient to enhance transfection, and 1 nM recombinant E3 enhanced the transfection 12-fold. Inhibitors of endocytosis suppressed this activity of the recombinant E3. These results suggest that the E3 fragment of Del1 can be used as a general biological enhancer of non-viral gene transfer.     

Turgor pressure regulation and the orientation of cortical microtubules in Spirogyra cells.

Microtubules (MTs) of cells of Spirogyra sp. were depolymerized by treatment with amiprophos-methyl (APM) for 1 h and then reorganized in 0.30 M mannitol solution. The reorganized MTs after 1.5 h incubation showed an oblique/longitudinal orientation and then became transversely oriented as the incubation was prolonged. During this incubation, the osmotic pressure of cells was measured by the plasmolysis method. The cell osmotic pressure increased with time. The calculated turgor pressure at 1.5 h was 0.11 M (mannitol equivalent) and, at 13.5 h, 0.25 M. Similar changes in MT orientation and recovery of the turgor pressure were also observed in 0.30 M sorbitol solution. These results suggest that the MT orientation may be correlated with the turgor pressure. Among fresh water algae sensitive to a saline environment, this Spirogyra was the first species shown to have a turgor regulating mechanism, although the recovery of turgor pressure was incomplete. The recovery of turgor pressure in mannitol solutions was also observed without APM treatment.     

Construction of a binary vector for transformation of Arabidopsis thaliana with a new selection marker

A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.     

Synthesis of 2-modified aristeromycins and their analogs as potent inhibitors against Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

2-Modified aristeromycin derivatives and their related analogs were synthesized to investigate their inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH). 2-Fluoroaristeromycin showed a strong inhibitory activity against PfSAHH selectively and complete resistance to adenosine deaminase.     

Potentiation of anti-aggregating activity of PGI2 by 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in rabbit platelets in vitro

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI₂, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID₅₀s of these agents in ADP-induced aggregation were 7–9 nM for PGI₂, 223 μM for EG-626, 266 μM for papaverine and 957 μM for dipyridamole. When EG-626 was used in combination with PGI₂, a threshold dose (50 μM) of EG-626 potentiated the anti-aggregating effect of subthreshold dose (3 nM) of PGI₂ upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI₂. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI₂ and EG-626 to induce 50% inhibition was obtained with 20% of ID₅₀ of each agent, whereas that of PGI₂ and papaverine or dipyridamole was 39 or 41%, respectively.     

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.     

Leaf senescence in rice due to magnesium deficiency mediated defect in transpiration rate before sugar accumulation and chlorosis

Magnesium (Mg) is an essential macronutrient supporting various functions, including photosynthesis. However, the specific physiological responses to Mg deficiency remain elusive. In this study, 2‐week‐old rice seedlings (Oryza sativa. cv. Nipponbare) with three expanded leaves (L2–L4) were transferred to Mg‐free nutrient solution for 8 days. In the absence of Mg, on day 8, L5 and L6 were completely developed, while L7 just emerged. We also studied several mineral deficiencies to identify specific responses to Mg deficiency. Each leaf was analyzed in terms of chlorophyll, starch, anthocyanin and carbohydrate metabolites, and only absence of Mg was found to cause irreversible senescence of L5. Resupply of Mg at various time points confirmed that the borderline of L5 death was between days 6 and 7 of Mg deficiency treatment. Decrease in chlorophyll concentration and starch accumulation occurred simultaneously in L5 and L6 blades on day 8. However, nutrient transport drastically decreased in L5 as early as day 6. These data suggest that the predominant response to Mg deficiency is a defect in transpiration flow. Furthermore, changes in myo‐inositol and citrate concentrations were detected only in L5 when transpiration decreased, suggesting that they may constitute new biological markers of Mg deficiency.     

Genetic diversity and the genetic structure of natural populations of Chamaecyparis obtusa: implications for management and conservation

We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H(e)) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G(ST)=0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F(ST)) and H(e) values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.     

Splicing factor SF3b as a target of the antitumor natural product pladienolide

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.     

Modulation of the biologic activities of IgE binding factor. VI. The activation of phospholipase by glycosylation enhancing factor

Glycosylation enhancing factor (GEF) from rat T cells is a kallikrein-like enzyme and enhances the assembly of N-linked oligosaccharides to IgE binding factors during their biosynthesis, whereas another T cell factor, i.e., glycosylation inhibiting factor (GIF), is a fragment of phosphorylated lipomodulin (i.e., phospholipase inhibitor), which when dephosphorylated inhibits phospholipase and the glycosylation process. The two T cell factors compete with each other when they are added to normal mesenteric lymph node cells during the formation of IgE binding factors. The addition of GEF to T cell hybridoma 23A4 cell switches the cells from the formation of unglycosylated IgE binding factor to the formation of N-glycosylated IgE binding factor. However, GEF neither inactivated GIF nor inhibited the formation of GIF by the T cell hybridoma. Stimulation of the T cell hybridoma with either affinity-purified GEF or bradykinin resulted in the release of GIF from the cells. GIF released by GEF stimulation had a m.w. of approximately 15,000 and bound to monoclonal antibody against lipomodulin. GEF and bradykinin also induced normal mesenteric lymph node cells to release GIF. Incorporation of 14C-arachidonic acid into 23A4 cells, followed by stimulation of the cells with GEF, resulted in the release of 14C-arachidonate. The results suggest that lipomodulin, a phospholipase inhibitory protein, is present in lymphocytes, and indicate that GEF and bradykinin induce the activation of phospholipase by stimulating cells to release lipomodulin.