Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically

Summary A comparative study was conducted to optimize the vegetative growth of sweet potato (Ipomoea batatas L. (Lam), cv. Beniazuma) plantlets cultured in vitro in five different types of supporting materials: agar matrix (a seaweed derivative; Kanto Chemical Co. Inc., Tokyo), gellan gum (a Pseudomonas derivative; Kanto Chemical Co. Inc., Tokyo), vermiculite (a kind of hydrous silicates), a mixture of vermiculite and cellulose fiber (Florialite; Nisshinbo Industries, Inc., Tokyo) and cellulose plug (Sorbarod; Baumgartner Rapiers SA., Switzerland). Single nodal cuttings were cultured photoautotrophically (without any sugar in the medium and with enriched CO₂ and high photosynthetic photon flux) for 21 d on MS basal medium. Plantlets exhibited the greatest growth when Florialite was used as supporting material. The leaf and root fresh and dry mass were 2.4× and 2.9×, and 2.2× and 2.8× greater, respectively, than those of the plantlets grown in the agar matrix (control). Plantlets cultured in Sorbarod supporting material exhibited the second greatest fresh and dry mass of leaves and roots followed by vermiculite and gellan gum supporting material. The most interesting feature was the development of a large number of fine lateral roots from the main adventitious root in the Florialite treatment. Among the treatments, the highest net photosynthetic rate was evident in the Florialite grown plantlets. The percent porosity of the supporting materials was highest in Sorbarod followed by Florialite and vermiculite. Plantlets transplanted from the Florialite supporting material exhibited the highest acclimatization percentage followed by that of the Sorbarod treatment.     

The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures

We previously reported that fetal rat calvaria (RC) cells are osteo-adipogenic bipotential and that PGE(2) receptors EP2 and EP4 are involved in bone nodule formation via both common and distinct MAPK pathways in RC cell cultures. Because PGE(2) participates in multiple biological processes including adipogenesis, it is of interest to determine the additional role(s) of PGE(2) in RC cells. PGE(2) increased the number of adipocyte colonies when RC cells were treated during proliferation but not other development stages. Of four EP agonists tested, the EP4 agonist ONO-AE1-437 (EP4A) was the most effective in promoting adipogenesis. Concomitantly, EP4A increased the number of cells with BrdU labeling and gene expression of CCAAT/enhancer binding protein (C/EBP)δ and c-fos but not peroxisome proliferator-activated receptor γ2 and C/EBPα. Amongst MAPK inhibitors, U0126, an inhibitor of MEK1/2, abrogated the EP4A-dependent effects. Our results suggest that the PGE(2)-EP4-ERK pathway increases the number of osteo-adipogenic bipotential progenitor cells, with a resultant increase in adipogenesis in RC cell cultures.     

Spectral quality and UV-B stress stimulate glycyrrhizin concentration of Glycyrrhiza uralensis in hydroponic and pot system

Glycyrrhizin, the major bioactive component of Glycyrrhiza uralensis, is widely used as a natural sweetener. Recently glycyrrhizin has been shown to have anti-tumor activity, highly active in inhibiting replication of HIV-1 and SARS-associated virus and exhibits a number of pharmacological effects. The principle objective of the current study was to evaluate the effects of different spectral quality including red, blue, white and UV-B radiation on the production of glycyrrhizin, in a controlled environment. Plants were grown under artificial lights with elevated CO(2) concentration and both the pot and hydroponic plants were assigned to red and blue light treatments and those grown under white fluorescent lamps were used as control. In a separate experiment, pot plants were exposed to ultraviolet (UV)-B radiation (wavelength: 280-315 nm). The net photosynthetic rates (NPR) of the leaves reduced significantly immediately after exposure to the high intensity UV-B radiation (3 days at 1.13 W m(-2)). In case of the low intensity UV-B radiation (15 days at 0.43 W m(-2)), NPR was also reduced, but the rate of reduction was significantly slower than that of the high intensity treatment. The concentrations of glycyrrhizin quantified in the root tissues were highest in the plants grown under red light in both hydroponic and pot systems and the concentration increased linearly from 1- to 3-month-old pot plants. Both the low and high intensity of UV-B exposure increased the concentration of glycyrrhizin in the root tissues of 3-month-old pot plants, the values being nearly X1.5 those of the control. The results also indicate that the glycyrrhizin concentrations of 3-6 months old pot plants were similar or even higher than the previously reported values for 3-4 years old field-grown plants and confirm that high concentration of glycyrrhizin production is possible within a very short production period under controlled environments.     

Intrafamilial distribution of mutans streptococci in Japanese families and possibility of father-to-child transmission

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.     

A new method for the synthesis of 2'-O-benzyladenosine using Mitsunobu reaction

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Mass Propagation of Eucalyptus camaldulensis in a Scaled-up Vessel Under In Vitro Photoautotrophic Condition

A scaled-up culture vessel was designed for the large-scalephotoautotrophic micropropagation of chlorophyllous plants.The culture vessel (volume 20 l) contained a plug cell traywith 448 plantlets, and had a forced ventilation system to supplyCO₂-enriched air. A nutrient-reservoir was connected to theculture vessel from which nutrient solution was circulated tothe culture vessel every 24 h. Nodal leafy cuttings of Eucalyptuscamaldulensis L. were cultured photoautotrophically in thissystem without sugar in the nutrient medium, but with an enrichedCO₂concentration and a high photosynthetic photon flux. Thegrowth and the net photosynthetic rate of the in vitro grownplantlets and the survival percentage of the plantlets aftertransplanting to ex vitro conditions were compared with thoseof plantlets grown photoautotrophically under natural ventilationin conventional small culture vessels (Magenta-type vessels;volume 0.4 l). Fresh and dry masses and net photosynthetic ratewere significantly higher in plantlets grown in the scaled-upvessel compared to plantlets grown in the conventional smallvessels (control). The environmental conditions created in thisscaled-up vessel (with forced ventilation) also facilitatedacclimatizationin vitro . Importantly, after transplanting tothe ex vitro condition, plantlets grew well without any specializedexvitro acclimatization treatment. Copyright 2000 Annals of BotanyCompany CO₂enrichment, Eucalyptus camaldulensis L., ex vitro, forced ventilation, natural ventilation, photoautotrophic, scaled-up vessel, survival percentage     

Water Control and Survival of Ipomoea batatas Grown Photoautotrophically under Forced Ventilation and Photomixotrophically under Natural Ventilation

Water loss and its determining factors were compared in leavesof in vitro -grown sweet potato (Ipomoea batatas L. Lam, ‘Beniazuma’)plantlets cultured either photoautotrophically under forcedventilation (scaled-up vessel) or photomixotrophically undernatural ventilation. Functional stomata, a greater amount ofwax deposition on the leaves and smaller epidermal conductancewere observed in plantlets cultured photoautotrophically underforced ventilation. When transplanted directly into soil inthe greenhouse (without ex vitro hardening), plantlets grownphotoautotrophically under forced ventilation survived betterand grew faster. This was because they were better able to controltranspiration and thus lost less water and showed no signs ofwilting. In contrast, plantlets cultured under photomixotrophicconditions had open stomata which were not functional, a highertranspiration rate, and uncontrolled and rapid water loss immediatelyafter transfer ex vitro. Thus, wilting and severe desiccationwith irreversible tissue damage were observed in the leaves,which died within a few days. Copyright 2000 Annals of BotanyCompany Epicuticular wax, hardening, stomata, sweet potato, transpiration, water control     

Pharmacokinetics and Efficacy of Topically Applied Nonsteroidal Anti-Inflammatory Drugs in Retinochoroidal Tissues in Rabbits

Purpose

To evaluate the pharmacokinetics and efficacy of topically applied nonsteroidal anti-inflammatory drugs (NSAIDs) in the retinochoroidal tissues of rabbits.

Methods

The cyclooxygenase (COX) inhibitory activity of diclofenac, bromfenac, and amfenac, an active metabolite of nepafenac, were determined using human-derived COX-1 and COX-2. Each of the three NSAIDs was applied topically to rabbits, and after 0.5 to 8 hrs, the concentration of each drug in the aqueous humor and the retinochoroidal tissues was measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetics of the drugs in the tissues after repeated doses as is done on patients was calculated by a simulation software. The inhibitory effect of each NSAID on the breakdown of the blood-retinal barrier was assessed by the vitreous protein concentration on concanavalin A-induced retinochoroidal inflammation in rabbits.

Results

The half-maximal inhibitory concentration (IC₅₀) of diclofenac, bromfenac, and amfenac was 55.5, 5.56, and 15.3 nM for human COX-1, and 30.7, 7.45, and 20.4 nM for human COX-2, respectively. The three NSAIDs were detected in the aqueous humor and the retinochoroidal tissue at all-time points. Simulated pharmacokinetics showed that the levels of the three NSAIDs were continuously higher than the IC₅₀ of COX-2, as an index of efficacy, in the aqueous humor, whereas only the bromfenac concentration was continuously higher than the IC₅₀ at its trough level in the retinochoroidal tissues. The intravitreous concentration of proteins was significantly reduced in rabbits that received topical bromfenac (P = 0.026) but not the other two NSAIDs.

Conclusions

Topical bromfenac can penetrate into the retinochoroidal tissues in high enough concentrations to inhibit COX-2 and exerts its inhibitory effect on the blood-retinal barrier breakdown in an experimental retinochoroidal inflammation in rabbits. Topical bromfenac may have a better therapeutic benefit than diclofenac and nepafenac for retinochoroidal inflammatory diseases.     

Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice

Background

Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.

Methods

To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.

Results

Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.

Conclusions

Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.     

Growth of In vitro banana (Musa SPP.) shoots under photomixotrophic and photoautotrophic conditions

Summary In vitro banana (Musa spp.) shoots were cultured under photomixotrophic (30 gl⁻¹ sucrose and 0.2 h⁻¹ number of air exchanges of culture vessels) and photoautotrophic (0 gl⁻¹ sucrose and 3.9 h⁻¹ number of air exchanges) conditions for 28 d in 370 cm³ Magenta boxes (GA7-type) containing 70 ml of half-strength Murashige and Skoog (MS) medium with 22.2 μM N⁶-benzyladenine (BA). The effects of varying CO₂ concentration (475 or 1340 μmol mol⁻¹) and light intensity (photosynthetic photon flux (PPF) of 100 or 200 μmol m⁻² s⁻¹) were investigated. Fresh and dry weights of banana shoots grown photomixotrophically were significantly greater on day 28 than those grown photoautotrophically. Photoautorophic shoots had a larger number of unfolded leaves and greater leaf area than photomixotrophic plants by days 14 and 28, regardless of CO₂ concentration. The shoot fresh and dry weights on day 14 in photoautotrophic conditions were significantly greater at PPF of 200 μmol m⁻² s⁻¹ than at 100 μmol m⁻² s⁻¹. The increase in net photosynthetic rate of photoautotrophic banana shoots was significant compared with photomixotrophic shoots. The multiplication ratio of in vitro banana shoots grown photoautotrophically in a 28-d culture period was the greatest at 100 μmol m⁻² s⁻¹ PPF and 475 μmol mol⁻¹ CO₂.