The Effects of Cytokinins on Unfolding of Cotyledons and Opening of Hypocotylar Hooks of Cucumber

Benzyladenine (BA), zeatin and kinetin caused the unfolding of cucumber (Cucumis sativus L. Var. Jing-ye No. 4) cotyledons. But the other plant hormones e.g. gibberellic acid (GA3), indoleacetic acid(IAA), ethylene (ethrel), abscisic acid (ABA) and other related compound e.g. KCl, adenine were not so. This response was caused by even lower concentration (0.01 ppm). Therefore, it could be used as a bioassay for cytokinins. KCl affaeted synergistically on cytokinin-induced unfolding. Basally applied BA (10 ppm) stimulated the opening of the intact hypocotylar hook. The light affacted synergistically on BA-induced opening. However, basally applied BA prevented the opening of decapitated hooks, especially in the light. The cotyledons were found to play a part of effects in maintaining the hook.     

The Effect of Actinomycin D and Cycloheximide on the Dedifferentiation of Cotyledon in Phaseolus radiatus L. and Its Nucleic Acid and Protein

Calli were induced from cotyledon segment of mung bean (Phaseolus radiatus L.) in Miller medium supplemented with NAA 4 mg/l, kinetin 10 mg/L. The callus formation was completely prevented by the addition of actinomycin D 15 μg/mL or cyclo- heximidc 0.5 μg/mL at 0 hour. The inhibitory effect of actinomycin D or cycloheximide was increased with the increment of concentration but decreased when the inhibitory agents were added a few hours later. If actinomycin D or cycloheximide was added at 24 hour culture it inhibits neither the induction of callus formation nor the proliferation. The content of RNA, DNA and protein were determined. RNA in each segment increased obviously in the early stage of callus formation, but DNA and protein increased slightly afterward. It is suggested that a large increase of RNA is the characteristic of dedifferentiation of cotyledon in P. radiatus. In addition, it has also been shown that an actinomycin D or cycloheximide-sensitive process in the early stage of dedifferentiation is crucial for the callus formation. Both RNA and protein synthesis are required for the initiation of dedifferentiation.     

Suppression and synthetic‐lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin‐binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.     

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan

Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibody's ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.     

Assessment of Phytotoxicity of Chromium in Flooded Soils using Embedded Selective Ion Exchange Resin Method

Chromium present in the forms of Cr(VI) or Cr(III) in soils. Since the toxicity and mobility of Cr(VI) are higher than those of Cr(III), it would be important to estimate soil Cr(VI) accurately in order to assess the phytotoxicity of Cr. Soil redox potential can influence the distribution of Cr between Cr(VI) and Cr(III) forms, and thus an in situ method which is not affected by the soil redox condition is needed for determining Cr(VI) availability in paddy fields. In this study, the Cu-saturated selective ion exchange resin (DOWEX M4159), serving as an infinite sink, was embedded in soils to extract available Cr(VI) from three representative saturated soils with different amounts of Cr(VI). The results suggested that Cr(VI) reduction occurred in the flooded soils, and the acid environment favored the adsorption and reduction of Cr(VI). There was a significant dose-response relationship between the soil resin-extractable Cr(VI) and the plant height of rice seedlings for test soils. The experimental results suggested that the embedded selective ion exchange resin method could be a suitable in situ method for assessing the phytotoxicity of Cr in flooded soils.     

Labyrinthulomycetes phylogeny and its implications for the evolutionary loss of chloroplasts and gain of ectoplasmic gliding

The labyrinthulomycetes, also known as the 'Labyrinthulomycota' are saprotrophic or less frequently parasitic stramenopilan protists, usually in marine ecosystems. Their distinguishing feature is an 'ectoplasmic net,' an external cytoplasmic network secreted by a specialized organelle that attaches the cell to its substrate and secretes digestive enzymes for absorptive nutrition. In this study, one of our aims was to infer the phylogenetic position of the labyrinthulomycetes relative to the non-photosynthetic bicoeceans and oomycetes and the photosynthetic ochrophytes and thereby evaluate patterns of change from photosynthesis to saprotrophism among the stramenopiles. For the labyrinthulomycetes, we determined sequences of the actin, beta-tubulin, and elongation factor 1-alpha gene fragments and where necessary, ribosomal small subunit (SSU) genes. Multilocus analysis using standard tree construction techniques not only strongly supported the oomycetes as the sister group to the phototrophic stramenopiles, but also, for the first time with moderate statistical support, showed that the labyrinthulomycetes and the bicoecean as sister groups. The paraphyly of the non-photosynthetic groups was consistent with independent loss of photosynthesis in labyrinthulomycetes and oomycetes. We also wished to develop a phylogenetically based hypothesis for the origin of the gliding cell bodies and the ectoplasmic net found in some labyrinthulomycetes. The cells of species in Labyrinthula and Aplanochytrium share a specialized form of motility involving gliding on ectoplasmic tracks. Before our study, only ribosomal DNA genes had been determined for these genera and their phylogenetic position in the labyrinthulomycetes was equivocal. Multilocus phylogenies applying our newly determined protein-coding sequences divided the labyrinthulomycetes between sister clades 'A' and 'B' and showed that the monophyletic group containing all of the gliding species was nested among non-gliding species in clade B. This phylogeny suggested that species that glide via an ectoplasm evolved from species that had used the ectoplasm mainly for anchorage and assimilation rather than motility.     

Characterization of microsatellite loci in the fungus, Grosmannia clavigera, a pine pathogen associated with the mountain pine beetle

The largest forest pest epidemic in Canadian history caused by the mountain pine beetle (MPB) and its fungal associates has killed over 15 million hectares of forest. Sixty simple sequence repeat regions were identified from Grosmannia clavigera, an MPB associated fungus. Eight loci genotyped in 53 isolates from two populations in British Columbia, Canada revealed three to 10 alleles per locus and gene diversities of 0 to 0.79. All but two of these loci showed length polymorphism in Leptographium longiclavatum, a related MPB fungal associate. These microsatellites will be useful in population genetic studies of these fungi.     

Genetic Diversity of the Cryptococcus Species Complex Suggests that Cryptococcus gattii Deserves to Have Varieties

The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV). To investigate the phylogenetic relationships among these haploid genotypes, we selected 73 strains from 2000 globally collected isolates investigated in our previous typing studies, representing each of these genotypes and carried out multigene sequence analyses using four genetically unlinked nuclear loci, ACT1, IDE, PLB1 and URA5. The separate or combined sequence analyses of all four loci revealed seven clades with significant support for each molecular type. However, three strains of each species revealed some incongruence between the original molecular type and the sequence-based type obtained here. The topology of the individual gene trees was identical for each clade of C. neoformans but incongruent for the clades of C. gattii indicating recent recombination events within C. gattii. There was strong evidence of recombination in the global VGII population. Both parsimony and likelihood analyses supported three major clades of C. neoformans (VNI/VNB, VNII and VNIV) and four major clades of C. gattii (VGI, VGII, VGIII and VGIV). The sequence variation between VGI, VGIII and VGIV was similar to that between VNI/VNB and VNII. MATa was for the first time identified for VGIV. The VNIV and VGII clades are basal to the C. neoformans or the C. gattii clade, respectively. Divergence times among the seven haploid monophyletic lineages in the Cryptococcus species complex were estimated by applying the hypothesis of the molecular clock. The genetic variation found among all of these haploid monophyletic lineages indicates that they warrant varietal status.     

CDK2/cyclinA inhibitors: targeting the cyclinA recruitment site with small molecules derived from peptide leads

The syntheses of potent small molecule inhibitors of the CDK2/cyclinA recruitment site are described. Structure-activity trends of nanomolar octapeptides were examined through amino-acid substitution and truncation of the sequence resulting in the identification of a smaller, albeit significantly less potent, tetrapeptide lead. These losses in affinity were recovered by side-chain optimization and by rigidification of the peptide backbone using a combination of solid-phase parallel synthesis and structure-based design. Finally, two guanidine functionalities were replaced to improve drug-like properties, resulting in neutral small molecules equal in activity to that of the peptide lead.