Changes of phenolic compounds in LebZIP2-overexpressing transgenic plants

484-491The bZIP gene is a transcription factor that plays various roles in relation to plant stress and hormone signaling. This gene is also involved in plant environmental stress and herbicide tolerance. We generated Nicotiana benthamiana transgenic plants with LebZIP2-encoding gene isolated from tomatoes using Agrobacterium-mediated transformation. Transgenic seeds harvested from these T0 transgenic plants were grown and examined for gene transfer and changes in phenolic compounds in the T1 generation. RT-PCR analysis using a primer specific to the LebZIP gene confirmed that the gene was transferred to the T1 generation. We analyzed the increase and decrease tendency for 30 phenolic compounds using the T1 generation-transgenic plants and investigated the mechanism between the specifically increased compound and LebZIP2 gene. Gallic acid, homogentisic acid, protocatechuic acid, myricetin, t-cinnamic acid, and b-resorcyclic acid were identified as the phenolic compounds that increased in T1 transgenic plants overexpressing the LebZIP gene. Among these, homogentisic acid at 246.75-1055.19 µg/g, was increased by 2-5 fold in the T1 transgenic plants compared to the control. Protocatechuic acid was found at 1640.54-2456.00 µg/g and was increased by 2-4 fold in T1 transgenic plants. t-Cinnamic acid was present in a small amount of 23.14 µg/g in the control, whereas it was 102.19-135.47 µg/g in T1 transgenic plants, showing an increase of 4-5 folds. These results indicated that homogentisic acid, protocatechuic acid, and t-cinnamic acid among the 30 phenolic compounds analyzed, were significantly increased in LebZIP2-overexpressing T1 transgenic plants, and support the evidence that the LebZIP2 gene is significantly involved in the increment of three phenolic compounds

Direct shoot organogenesis from petiole and leaf discs of Withania somnifera (L.) Dunal

An efficient and reproducible procedure is described for direct shoot regeneration using petiole and leaf explants of Withania somnifera (L.). The shoots were mainly induced from the distal end of the petiole, whereas in leaf explants, shoot regeneration was initiated from the basal part and wounded tissue. The regeneration medium that induced the highest numbers of shoots in the petiole and leaf explants was Murashige and Skoog (MS) medium supplemented with 2 mg/l N6-benzyladenine (BA) alone or with 0.1 mg/l a-naphthalene acetic acid (NAA). The frequency of shoot regeneration was greatly influenced by the type of explant, the carbon source, the orientation of the explant, and the basal medium used in the regeneration medium. Explants produced shoot buds and adventitious shoots within four weeks. Histological analysis of the regenerating shoots showed that the shoot buds emerged from sub epidermal parenchymal cells, with no intermediate callus formation. Plantlets were rooted on MS alone or MS containing different concentrations of 3-indolebutyric acid (IBA). The addition of 1 mg/l IBA to the medium was most effective in inducing root formation. The regenerated plantlets were acclimatized in the greenhouse and successfully transferred to the field, with a 90% survival rate. The acclimatized plants showed normal flowering and were not morphologically different from the seed-derived mother plants.Key words: Histology, medicinal plant, plant growth regulator, plant regeneration, Withania somnifera

Valproic Acid Downregulates the Expression of MGMT and Sensitizes Temozolomide-Resistant Glioma Cells

Temozolomide (TMZ) has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. Valproic acid (VPA) has emerged as an anticancer drug via inhibition of histone deacetylases (HDACs), but the therapeutic advantages of a combination with VPA and TMZ remain poorly understood. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of VPA and TMZ, especially in TMZ-resistant cell lines. A combination of VPA and TMZ had a significantly enhanced antitumor effect in TMZ-resistant malignant glioma cells (T98 and U138). This enhanced antitumor effect correlated with VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, which plays an important role in cellular resistance to alkylating agents. In vitro, the combination of these drugs enhanced the apoptotic and autophagic cell death, as well as suppressed the migratory activities in TMZ-resistant cell lines. Furthermore, in vivo efficacy experiment showed that treatment of combination of VPA and TMZ significantly inhibited tumor growth compared with the monotherapy groups of mice. These results suggest that the clinical efficacy of TMZ chemotherapy in TMZ-resistant malignant glioma may be improved by combination with VPA

Tat peptide-admixed elastic liposomal formulation of hirsutenone for the treatment of atopic dermatitis in NC/Nga mice

Myung Joo Kang1, Jae Yoon Eum1, Mi Sook Jeong2, Sang Han Park1, Ki Young Moon1, Mean Hyung Kang1, Min Soo Kim1, Sun Eun Choi1, Min Won Lee1, Do Ik Lee1, Hyoweon Bang2, Chung Soo Lee2, Seong Soo Joo3, Kapsok Li2, Mi-Kyung Lee2, Seong Jun Seo2, Young Wook Choi11College of Pharmacy, ChungAng University, Heuksuk-dong, Dongjak-gu, Seoul, 2College of Medicine, Chung-Ang University, Heuksukdong, Dongjak-gu, Seoul, 3Division of Marine Molecular Biotechnology, Gangneung-Wonju National University, Gangneung, South KoreaBackground: The aim of the present study was to enhance a topical delivery of hirsutenone (HST), a naturally occuring immunomodulator, employing Tat peptide-admixed elastic liposomes (EL/T).Methods: HST-loaded EL, consisting of phosphatidylcholine and Tween 80 (85:15 w/w%), were prepared using thin film hydration method. By adding Tat peptide to EL (0.16 w/w%), EL/T were formulated. The in vitro skin permeation of HST was examined using a Franz diffusion cell mounted with depilated mouse skin. Lesions for atopic dermatitis (AD) were induced by a topical application of diphenylcyclopropenone to NC/Nga mice. Therapeutic improvements of AD were evaluated by clinical skin severity scores. Immunological analyses on inducible nitric oxide synthase and cyclooxygenase-2 levels in the skin and interleukin (IL)-4, IL-13, immunoglobulin E, and eosinophil levels in the blood were also performed.Results: EL systems were superior to conventional cream, revealing greater flux values in a permeation study. The addition of Tat peptide further increased the skin permeation of HST. In an efficacy study with AD-induced NC/Nga mice, an HST-containing EL/T formulation brought a significant improvement in both skin severity score and immune-related responses for the levels of nitric oxide synthase, cyclooxygenase-2, IL-4, IL-13, immunoglobulin E, and eosinophils.Conclusion: A novel EL/T formulation was successfully developed for topical delivery of HST to treat AD.Keywords: hirsutenone, elastic liposomes, atopic dermatitis, NC/Nga mice, Tat peptid

Comparison of anterior segment optical coherence tomography findings in acanthamoeba keratitis and herpetic epithelial keratitis

This study is to investigate the characteristic features of Acanthamoeba keratitis (AK) that differentiating it from herpetic epithelial keratitis (HEK) using anterior segment optical coherence tomography (AS-OCT). Medical records of three eyes of each AK and herpetic keratitis who had AS-OCT examination were reviewed in this study. Slit-lamp biomicroscopy and AS-OCT was performed on the initial visit and on every follow-up visits in all patients. In all three AK cases, reflective bands in the corneal stroma that correspond to the area of radial keratoneuritis were observed. The depth of the reflective bands varied in each case. After AK treatment, slit-lamp biomicroscopy confirmed that radial keratoneuritis had resolved and AS-OCT confirmed that reflective bands in the corneal stroma had also disappeared in all patients. Unlike the AS-OCT results found in AK, highly reflective HEK lesions were observed only in the subepithelial area, not in the stroma. AS-OCT seems to be helpful analyzing the specific depth of the lesion which enables to distinguish AK from HEK

Atopic Dermatitis-Like Skin Lesions Reduced by Topical Application and Intraperitoneal Injection of Hirsutenone in NC/Nga Mice

Atopic dermatitis (AD) is a common inflammatory skin disease. The increasing prevalence and severity of AD have prompted the developments of safer, more effective drugs. Although topical corticosteroids have been used as first line therapy for AD, their potential side effects limit their clinical applications. To investigate the effect of hirsutenone (HIR), a diarylheptanoid compound, on AD-like skin lesions and other factors related to immune response is the aim of this paper Th2-related cytokines (IL-4, IL-5, IL-13), eosinophil, IgE inflammatory factors (COX-2, iNOS) levels were reduced in blood, lymphocytes, and tissue after HIR treatment. These results suggest that HIR might be an effective treatment for AD