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 Scutellaria ocmulgee (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/ß 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/ß 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.

Development of monoclonal antibodies against bovine mucin core 2 beta6 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 beta6N-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(-1)) for these MAbs range from 5.4 x 10(7) to 1.2 x 10(9). 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.

Isolation and characterization of a water stress-specific genomic gene, pwsi 18, from rice.

One of the water stress-specific cDNA clones of rice characterised previously, wsi18, was selected for further study. The wsi18 gene can be induced by water stress conditions such as mannitol, NaCl, and dryness, but not by ABA, cold, or heat. A genomic clone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstream sequence, two introns, and the full coding sequence. The 5'-upstream sequence of pwsi18 contained putative cis-acting elements, namely an ABA-responsive element (ABRE), three G-boxes, three E-boxes, a MEF-2 sequence, four direct and two inverted repeats, and four sequences similar to DRE, which is involved in the dehydration response of Arabidopsis genes. The gusA reporter gene under the control of the pwsi18 promoter showed transient expression in response to water stress. Deletion of the downstream DRE-like sequence between the distal G-boxes-2 and -3 resulted in rather low GUS expression.

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 microM, 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 wsi18 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.