Nirmal Joshee

Induction of chilling resistance by water stress, and cDNA sequence analysis and expression of water stress-regulated genes in rice

Exposure of seedlings of a chilling-sensitive variety of rice (Oryza sativa L. cv. Wasetoittu) to water stress (0.5 M mannitol, 30 min) at room temperature induced a degree of chilling resistance. No such resistance was induced by exogenous abscisic acid (ABA) application (10 μM, 60 min). Upon short-term water stress, new transcripts were expressed in both seedlings and suspension-cultured cells. We suggest that the genes induced by short-term water stress, and not those induced by ABA, are related to acquired chilling resistance in this chilling-sensitive rice variety. A total of nine different cDNA clones, specifically induced by short-term water stress, were isolated by differential hybridization and partial sequencing. Northern hybridization analysis using RNAs from the seedlings subjected to chilling after water stress treatment reveal three distinct groups of above mentioned nine cDNA clones: wsi (water stress-induced) 18, 76, and 724, representative of genes whose expression increases, decreases, and remains almost fixed during chilling, respectively. The nucleotide and deduced amino acid sequences of the three representative clones were determined. Characteristic features of wsi 18 are the presence of one set of amino acid sequence repeats, a conserved amino acid sequence common to LEA-group genes in the N-terminal region, and an alanine- and lysine-rich tract in the C-terminal region.

In vitro antitumor mechanisms of various Scutellaria extracts and constituent flavonoids

Scutellaria is a traditional herbal remedy with potential anti-cancer activity. The purpose of this study was to evaluate anticancer mechanisms of thirteen Scutellaria species and analyze their leaf, stem and root extracts for levels of common biologically active flavonoids: apigenin, baicalein, baicalin, chrysin, scutellarein, and wogonin. Malignant glioma, breast carcinoma and prostate cancer cells were used to determine tumor-specific effects of Scutellaria on cell proliferation, apoptosis and cell cycle progression, via the MTT assay and flow cytometry-based apoptosis and cell cycle analysis. The extracts and individual flavonoids inhibited the proliferation of malignant glioma and breast carcinoma cells without affecting primary or non-malignant cells. The flavonoids exhibited different mechanisms of anti-tumor activity as well as positive interactions. The antitumor mechanisms involved induction of apoptosis and cell cycle arrest at G1/G2. Of the extracts tested, leaf extracts of S. angulosa, S. integrifolia, S. ocmulgee and S. scandens were found to have strong anticancer activity. This study provides basis for further mechanistic and translational studies into adjuvant therapy of malignant tumors using Scutellaria leaf tissues.

Transferrin-Facilitated Lipofection Gene Delivery Strategy: Characterization of the Transfection Complexes and Intracellular Trafficking

We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.

Delayed growth of glioma by Scutellaria flavonoids involve inhibition of Akt, GSK-3 and NF-κB signaling

Plants of the genus Scutellaria constitute one of the common components of Eastern as well as traditional American medicine against various human diseases, including cancer. In this study, we examined the in vivo anti-glioma activity of a leaf extract of Scutellariaocmulgee (SocL) while also exploring their potential molecular mechanisms of action. Oral administration of SocL extract delayed the growth of F98 glioma in F344 rats, both in intracranial and subcutaneous tumor models. Immunohistochemistry revealed inhibition of Akt, GSK-3α/β and NF-κB phosphorylation in the subcutaneous tumors following treatment with Scutellaria. The SocL extract as well as the constituent flavonoid wogonin also showed dose- and time-dependent inhibition of Akt, GSK-3α/β and NF-κB in F98 cell cultures in vitro, as determined by western blot analysis. Pharmacologic inhibitors of PI3K and NF-κB also significantly inhibited the in vitro proliferation of F98 glioma cells, indicating the key role of these signaling molecules in the growth of malignant gliomas. Transfection of F98 cells with constitutively active mutant of AKT (AKT/CA), however, did not significantly reverse Scutellaria-mediated inhibition of proliferation, indicating that Scutellaria flavonoids either directly inhibited Akt kinase activity or acted downstream of Akt. In vitro Akt kinase assay demonstrated that the SocL extract or wogonin could indeed bind to Akt and inhibit its kinase activity. This study provides the first in vivo evidence and mechanistic support for anti-glioma activity of Scutellaria flavonoids and has implications in potential usage of Scutellaria flavonoids in adjuvant therapy for malignant tumors, including gliomas.

Effect of light, methyl jasmonate and cyclodextrin on production of phenolic compounds in hairy root cultures of Scutellaria lateriflora

Scutellaria lateriflora (American skullcap) has been used in traditional medicine to treat several medical conditions including nervous disorders and cancer. Previous studies have associated these medicinal properties to flavones present in roots and leaves of this species. In order to develop a production system and study the biosynthesis of these bioactive compounds, hairy root cultures of S. lateriflora were established and line 4 was selected for further studies based on its growth performance in a modified Murashige and Skoogs medium supplemented with 0.5mg/l indole-3-butyric acid. Scanning electron microscopy of the hairy roots showed a high profusion of hairs along the root. Several phenolic compounds, including verbascoside, and the flavones wogonin, baicalein, scutellarein and their respective glucuronides were identified by high performance liquid chromatography-tandem mass spectrometry in the root tissue, but not in the culture medium. Among these compounds, verbascoside accumulated at the highest levels. Interestingly, cultures incubated under continuous light and treated with 15mM methyl-β-cyclodextrin for 24h produced significantly higher levels of the aglycones, baicalein and wogonin, but not scutellarein, compared to cultures incubated under continuous darkness. This work demonstrates that hairy root cultures of S. lateriflora have the biosynthetic capacity to produce known Scutellaria flavones and suggest that light may have a selected regulatory effect on the synthesis or accumulation of these phenolic compounds.

Scutellaria extract and wogonin inhibit tumor-mediated induction of T reg cells via inhibition of TGF-β1 activity

A number of studies have implicated tumor-induced Treg cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting Treg cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced Treg activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and Treg cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated Treg activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in Treg culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in Treg cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells’ response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells’ response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

Scutellaria: Biotechnology, Phytochemistry and Its Potential as a Commercial Medicinal Crop

Plants of the genus Scutellaria (family Lamiaceae) are distributed globally and they are integral part of Eastern as well as American traditional medicine. The genus Scutellaria, commonly referred to as skullcap, is considered as a North American perennial plant. At present this genus is represented by 350–360 species. Many species are rare, threatened, or endangered. Habitat destruction, urbanization, and poor seed set are a few reasons behind the diminishing population of many skullcaps. Many skullcap species have showy, beautiful blooms with great potential as ornamental plants. Skullcaps are used in alternative medicine as antiinflammatory, antispasmodic, emmenagogue, nervine, sedative, and strong tonic. More than 295 compounds have been isolated from Scutellaria species and the majority of the compounds are flavonoids and diterpenes. We present a review of research carried out by various groups, covering aspects of phytochemical screening, biomedical studies, conservation, plant biotechnology, and development of Scutellaria as a premium crop. We have developed a germplasm collection at Fort Valley State University that includes 19 species. These species are maintained in the greenhouse and through micropropagation in the lab. Biotechnology presents significant advances for the use of Scutellaria. We will review progress in micropropagation, transformation for desired gene transfer, and hairy root induction, extraction, and HPLC analysis of targeted flavonoids, and preclinical and clinical studies on select extracts and isolated flavonoids using various cancer models.

Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species

The antioxidant capacity of 16 Scutellaria species was examined using Trolox equivalent antioxidant capacity (TEAC) assay. Total polyphenol, antioxidant capacity estimation and flavonoid content measurements were conducted on fresh and air dried leaf extracts. The highest total polyphenol content was obtained in dry leaf extracts of S. ocmulgee at 732.41 67 mg/g of gallic acid equivalent. Dry leaf extracts of S. ocmulgee registered 2480.93 μmol/g of Trolox equivalent antioxidant capacity and the highest total flavonoid content with 200.63 μg/mL from fresh leaf extracts of S. alpina. Rosemary (Rosmarinus officinalis), a common herb with known high antioxidant potential, was used as a standard for comparison with the Scutellaria species. INTRODUCTION The genus Scutellaria, commonly known as skullcap (scullcap) belongs to the family Lamiaceae (Mint family). Of the 400 known Scutellaria species, over 90 of these plants have been recorded as growing in North America. In the current study, 20 Scutellaria spp. that grow in and adjacent to the state of Georgia were used (Joshee et al., 2002). Scutellaria ocmulgee and Scutellaria montana, two species that grow in Georgia, are relatively rare and protected by the state and federal government (Chafin, 2007). Scutellaria species are known for their potential pharmaceutical/therapeutic properties and have been used in the traditional medicine of many countries for their anti-inflammatory, antioxidant, and anti-viral properties useful in treating human ailments (Huang et al., 2005b; Shang et al., 2010). S. baicalensis, native to China, and S. barbata, from Korea, have been extensively employed in traditional Chinese medicine (TCM) and in Japanese Kampo medicine (JKM) (Watanabe et al., 2002; Murch et al., 2004). Leaf extract from S. ocmulgee has demonstrated inhibitory properties against malignant gliomas (Parajuli et al., 2009, 2011). Plants, such as basil (Ocimum spp.), mint (Mentha spp.), rosemary (Rosmarinus officinalis), lavender (Lavandula spp.), and Baikal skullcap (Scutellaria baicalensis), are known to contain relatively high levels of phenolics and have demonstrated antioxidant activity (Zheng and Wang, 2001; Shao et al., 2004; Waisundara, 2010; Atanassova and Georgieva, 2010). Leaves from peppermint (M. x piperita), rosemary, sage (Salvia spp., spearmint (M. spicata), and thyme (Thymus spp. growing in a greenhouse exhibit total polyphenol (TPP) and Trolox equivalent antioxidant capacity (TEAC) as plants grown under field conditions, plus anti-tumorigenic activity against colon cancer cells (Yi and Wetzstein, 2010; 2011). Free radicals can cause disorders, such as atherosclerosis, central nervous system injury, and gastritis in the human body (Kumpulainen and Salonen, 1999; Pourmorad et al., 2006). By boosting the human immune system, plant-based antioxidants block free radicals produced through oxidation (Schuler, 1990), thus inhibiting chain reactions that could lead to degradation and death of cells (Pratt, 1992; Velioglu et al., 1998; Hu and Willett, 2002). The antioxidant activity delays and inhibits oxidation of cellular components and molecules (Nijveldt et al., 2001). Thus, the level of activity of various phytochemicals is important for the evaluation of their potential health benefit to humans. Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

Freezing tolerance and cold acclimation in guava (Psidium guajava L.)

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Development of monoclonal antibodies against bovine mucin core 2 β6 N-acetylglucosaminyltransferase

Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 β6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M−2) for these MAbs range from 5.4 × 107 to 1.2 × 109. These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, These antibodies should be useful for further study of bovine and human core 2 enzymes.