Lepidimoide Promotes Light-Induced Chlorophyll Accumulation in Cotyledons of Sunflower Seedlings

The effect of disaccharide lepidimoide on light-induced chlorophyll accumulation was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings and detached cucumber (Cucumis sativus L.) cotyledons. From studies on the structure-activity relationships of lepidimoide, its analogs, and sucrose with respect to light-induced chlorophyll accumulation in the cotyledons of sunflower seedlings, both lepidimoide and the free carboxylic acid of lepidimoide (lepidimoic acid) showed the highest promoting activity, whereas the hydrogenated lepidimoide, which lacks a double bond in the C4, 5 position in uronic acid, showed lower activity than lepidimoide; however, sucrose exhibited very weak activity. These results suggest that lepidimoide acts as a new type of plant growth regulator, not simply as a carbon source providing energy. Lepidimoide promoted not only light-induced chlorophyll accumulation in sunflower cotyledons but also light-induced 5-aminolevulinic acid content, which is considered to be a rate-limiting step in chlorophyll biosynthesis. Lepidimoide with cytokinin stimulated the accumulation of chlorophyll and 5-aminolevulinic acid additively. In detached cucumber cotyledons, lepidimoide also promoted light-induced chlorophyll accumulation. These results indicate that lepidimoide, in cooperation with cytokinin, causes light-induced chlorophyll accumulation in the cotyledons of several dicot plant species by affecting the level of 5-aminolevulinic acid. Received April 4, 1997; accepted September 28, 1998     

Mass propagation of ornamental gentian in liquid medium

An efficient system for clonal mass propagation in liquid culture was established for the propagation of ornamental gentian. In a test of the requirements for three cytokinins [6-benzylaminopurine, N-(2-chloro-4-pyridyl)-N′-phenylurea and N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ)] in combination with 1-naphthaleneacetic acid (NAA), we found that effective propagation of shoots occurred with 0.01 mg l^–1 TDZ in a 300 ml conical flask that contained 100 ml of medium. The propagation of shoots was also affected by the concentrations of macronutrients (KNO₃, NH₄NO₃ and CaCl₂) and sucrose in Murashige and Skoog's (MS) medium, and it was influenced to some extent by the speed of agitation on an orbital shaker. The most efficient propagation of shoots was achieved in full-strength MS medium supplemented with 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose with agitation at 150 rpm. The propagation of shoots was maximal after 6 weeks of culture (140 shoots from five nodal segments in one flask). Large-scale propagation in a 5-l fermenter was attempted using 3 l of MS medium that contained 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose. More than 2,000 shoots were obtained in the fermenter in 5 weeks following the initial cultivation of five nodal segments for 6 weeks in one 300-ml flask. The shoots that had propagated in the fermenter were transferred directly to soil without prior rooting in vitro and were easily acclimatized within 1 month. Received: 7 October 1997 / Revision received: 16 January 1998 / Accepted: 30 January 1998     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.     

How to compute the effective size of spatiotemporally structured populations using separation of time scales

Calculations to derive effective population size become highly complicated when complex population structure is considered. We provide an easy method of computing the effective size of a subdivided population with overlapping generations (a spatiotemporally structured population) using an approximation based on separation of time scales. We also numerically compute the effective size to verify the accuracy of the derived formula. Various interesting quantities, including moments of coalescent time, are readily derived using this approach.     

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vasculitis induced by immunization with Bacillus Calmette-Guérin followed by atypical mycobacterium antigen: a new mouse model for Kawasaki disease

Kawasaki disease causes systemic vasculitis. The development of skin lesions at the vaccination site with Bacillus Calmette-Guérin (BCG) is an important diagnostic symptom. We hypothesized that infection with ubiquitous microorganisms immunogenically related to BCG might induce an immunopathologic reaction leading to the development of Kawasaki disease. Mice were first inoculated with BCG, and then secondarily inoculated 4 weeks later with crude extract from Mycobacterium intracellulare (cMI), an abundant atypical mycobacterium. Animals inoculated with BCG followed by cMI developed coronary arteritis with infiltration of inflammatory cells, whereas control animals inoculated with only cMI or BCG did not, suggesting that the immune response to the mycobacteria induced autoimmunity to the vascular wall. Intravenous injection with antibodies to peroxiredoxin II, a modulator of vascular remodeling and a suggested target for autoimmune vasculitis, also resulted in coronary arteritis, but only after prior inoculation with BCG. Tumor necrosis factor-alpha, MCP1 and interferon-gamma production were significantly higher in the animals inoculated with BCG than in the control groups (P<0.05). BCG immunization was required for the development of coronary arteritis, suggesting that these cytokines might play important roles. The results indicate that BCG induces primary autoimmunity and stimulates cytokine induction, and that atypical mycobacterial infection boosts the autoimmunity resulting in coronary arteritis.     

The floor plate is sufficient for development of the sclerotome and spine without the notochord

Danforth'sshort-tail (Sd) mouse is a semi-dominant mutation affecting the development of the vertebral column. Although the notochord degenerates completely by embryonic day 9.5, the vertebral column exists up to the lumber region, suggesting that the floor plate can substitute for notochord function. We previously established the mutant mouse line, Skt(Gt), through gene trap mutagenesis and identified the novel gene, Skt, which was mapped 0.95cM distal to the Sd locus. Taking advantage of the fact that monitoring notochordal development and genotyping of the Sd locus can be performed using the Skt(Gt) allele, we assessed the development of the vertebra, notochord, somite, floor plate and sclerotome in +-+/+-Skt(Gt), Sd-+/+-+, Sd-Skt(Gt)/+-+, Sd-Skt(Gt)/+-Skt(Gt), Sd-+/Sd-+ and Sd-Skt(Gt)/Sd-Skt(Gt) embryos. In Sd homozygous mutants with a C57BL/6 genetic background, the vertebral column was truncated in the 6th thoracic vertebra, which was more severe than previously reported. The floor plate and sclerotome developed to the level of somite before notochord degeneration and the number of remaining vertebrae corresponded well with the level of development of the floor plate and sclerotome. Defects to the sclerotome and subsequent vertebral development were not due to failure of somitogenesis. Taken together, these results suggest that the notochord induced floor plate development before degeneration, and that the remaining floor plate is sufficient for maintenance of differentiation of the somite into the sclerotome and vertebra in the absence of the notochord.     

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.