Sadanandam Abbagani

A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae).

INTRODUCTION It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. OBJECTIVE To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). METHODOLOGY Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. RESULTS The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT-PCR analysis. CONCLUSION The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from various recalcitrant Euphorbiaceae members.

Indolylmethylene benzo[h]thiazolo[2,3-b]quinazolinones: Synthesis, characterization and evaluation of anticancer and antimicrobial activities

A series of novel 10-((1H-indol-3-yl)methylene)-7-aryl-7,10-dihydro-5H-benzo[h]thiazolo[2,3-b]quinazolin-9(6H)-ones (8a-t) have been synthesized in good yields by the reaction of benzo[h]quinazoline-2(1H)-thiones (4a-f) with 2-chloro-N-phenylacetamide (5) followed by Knoevenagel condensation with various indole-3-carbaldehydes (7a-d) under conventional method. All the synthesized compounds were characterized by spectral studies and screened for their in vitro anticancer and antimicrobial activities. Compound 8c has exhibited excellent activity against MCF-7 (breast cancer cell line) than the standard drug Doxorubicin. Compound 8d against both the cancer cell lines, 8q against MCF-7 and 8c, 8h against HepG2 have also shown good activity. Remaining compounds have shown moderate activity against both the cell lines. Antimicrobial activity revealed that, the compound 8q and 8t against Staphylococcus aureus and 8i, 8k, 8l, 8q &8t against Klebsiella pneumoniae have shown equipotent activity on comparing with the standard drug Streptomycin. Remaining compounds have shown significant antibacterial and comparable antifungal activities against all the tested microorganisms.

Pterostilbene as a potential novel telomerase inhibitor: molecular docking studies and its in vitro evaluation.

Pterostilbene is a naturally occurring dimethyl ether analog of resveratrol identified in several plant species. Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. Molecular docking studies were performed on pterostilbene with the crystal structure of telomerase (3DU6). Pterostilbene was evaluated for its in vitro cytotoxicity in breast (MCF7) and lung cancer (NCI H-460) cell lines, antimitotic activity in green grams and telomerase activity. Curcumin was used as a standard. Docking results indicated good interaction between pterostilbene and the active site of telomerase and the docked energy of pterostilbene was -7.10 kcal/mol. Pterostilbene showed strong inhibitory effect on in vitro telomerase activity and cell growth in both the cell lines tested in a dose dependent manner. Cancer cells treated with 80 µM pterostilbene exhibited significant telomerase inhibition, after 72 hours (MCF-7 and NCI H-460; 81.52% and 74.69% reduction, respectively, compared to control). The IC50 of pterostilbene for anti-proliferative activity in MCF7 and NCI H-460 cell lines were found to be 30.0 and 47.2 µM, respectively. The best antimitotic activity was obtained with 80 μM of pterostilbene (100% reduction in water imbibition). All the above results were comparable to that of curcumin. The drug-related properties of pterostilbene were calculated using Molinspiration, Osiris Property Explorer and ACD/Chemsketch softwares. Pterostilbene obeyed Lipinskis Rule of Five indicating its therapeutic potential in humans. It was found that the telomerase inhibitory activity exhibited by pterostilbene was dependent of the cell viability and has the potential to be a new drug candidate against breast and lung cancers.

Synthesis, characterization and biological evaluation of fused thiazolo[3,2-a]pyrimidine derivatives

A series of fused thiazolo[3,2-a]pyrimidines (7a–g, 8a–f, 11a–g and 12a,b) have been synthesized in good yields by reaction of fused 3,4-dihydropyrimidin-2(1H)-thiones (4a–g) with phenacyl bromides (5,6)/3-(2-bromoacetyl)coumarins (9,10) under conventional heating in acetic acid. Analytical and spectral studies as well as single crystal X-ray diffraction data on the representative compound 8e confirmed the structure of all the reaction products. All the synthesized compounds were screened for their antibacterial, antioxidant and DNA cleavage activities. The compound 7e against Escherichia coli, 8a and 8c–e against Pseudomonas aeruginosa have shown prominent antibacterial activity compared to the standard drug Penicillin with MIC 9.375 μg mL−1, whereas the compounds 11c, 12a and 12b have shown very good antioxidant activity compared to the standard drug Trolox with IC50 values 12.36, 11.12 and 13.88 μM respectively. Compounds 11f and 12b have completely cleaved the DNA even at 50 μg mL−1 concentration and the remaining compounds have partially cleaved the DNA.

Efficient In-Vitro Regeneration from Mature Leaf Explants of Scoparia dulcis L., an Ethnomedicinal Plant

ABSTRACT A plant regeneration protocol via multiple-shoot induction using leaf explants from field-grown mature plants of Scoparia dulcis L. (Scrophulariaceae), an ethnomedicinal plant, was established. Explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP, 13.2, 17.6 and 22.2 μM) in combination with indole-3-acetic acid (IAA, 0.5 and 2.8 μM) or napthalene-3-acetic acid (NAA, 0.5 and 2.6 μM). The maximum number of shoots (14.0 ± 1.14) with longest shoot length (2.97 ± 0.18) were obtained directly (without an intervening callus phase) from the leaf explants using a combination of BAP (22.2 μM) and IAA (0.5 μM). BAP used in combination with NAA produced fewer shoots per explant as compared with the use of IAA and the regeneration was mediated by callus formation. For root induction, the elongated shoots were separated and transferred onto MS medium supplemented with indole-3-butyric acid (IBA, 4.9 μM). Rooted plantlets (90%) were successfully transferred to soil in the greenhouse with a 75% survival rate.

Evaluation of serum human telomerase reverse transcriptase as a novel marker for cervical cancer.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of human telomerase and its rate-limiting component. The purpose of the present study was to investigate the diagnostic value of hTERT in serum of cervical cancer patients. Preoperative values of hTERT, squamous cell carcinoma antigen (SCC-ag) and cancer antigen 125 (CA 125) were measured by enzyme-linked immunosorbent assay (ELISA) in 192 patients with squamous cell carcinoma or adenocarcinoma of the uterine cervix and 38 healthy controls. Elevated pretreatment levels of hTERT were identified in 80.2% of squamous cell carcinoma and 73.8% of adenocarcinoma patients. The expression of serum hTERT was correlated with telomerase activity in cancer tissues of both histological types. Pretreatment serum hTERT levels showed a significant correlation with clinical stage, tumor size and lymph node metastasis, but not with age. Serum hTERT measurement was found to be useful in the diagnosis and assessment of clinical stage of cervical cancer, and to be superior to the conventional tumor markers. Therefore, serum hTERT is a novel and readily available marker for cervical malignancies.

Antioxidant and Analgesic Activities of Pterocarpus marsupium Roxb

The methanolic extract of the bark of Pterocarpus marsupium Roxb. (Papilionaceae) was screened for antioxidant activity in vitro and analgesic activity in mice, respectively. The in vitro antioxidant activity of the bark extract was evaluated using the 1,1-diphenyl-2-picrylhydrazyl assay, and the results were expressed as IC50. Ascorbic acid, used as a standard, had an IC50 = 34.0 μg mL−1, whereas the bark extract of P. marsupium had an IC50 = 53.0 μg mL−1. Use of the hot-plate method to study central analgesic activity of the bark extract in mice indicated that the bark extract of P. marsupium possesses the ability to significantly reduce pain threshold and also increase the response latency period to thermal stimuli in mice, similar to the reference drug pentazocine. The reaction time of mice was significantly increased to 2 hr with 500 mg mL−1 of bark extract, whereas pentazocine also increased reaction time to 2 hr with 5 mg kg−1. A decline in the reaction time beyond 3 hr was observed by the reference drug and bark extract.

Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na+/H+ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T1 generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H2O2), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.

Serum human telomerase reverse transcriptase: a novel biomarker for breast cancer diagnosis.

BackgroundTelomerase is a ribonucleoprotein complex composed mainly of a reverse transcriptase catalytic subunit, telomerase reverse transcriptase (hTERT). Expression of hTERT confers telomerase activity, indicating that hTERT is the rate-limiting component of human telomerase. The aim of the present study was to investigate the diagnostic implications of hTERT in the serum of breast cancer patients.MethodsThe study was conducted on 159 breast cancer patients and 41 healthy volunteers as controls. The evaluation of hTERT, cancer antigen 15.3 and carcinoembryonic antigen were performed by enzyme-linked immunosorbent assay, and analysed for their correlation with the patient’s clinicopathological features.Results27 of 52 (51.9%) patients with stage I breast cancer, 31 of 40 (77.5%) with stage II and 30 of 34 (88.2%) patients with stage III exhibited elevated hTERT levels. Serum hTERT levels showed significantly higher mean values in patients with breast cancer than healthy individuals. The sensitivity and specificity of hTERT in cancer diagnosis was 68.9 and 83.3%, respectively, which is significantly higher than conventional markers. The expression of serum hTERT was significantly correlated with telomerase activity in breast cancer tissues. Pretreatment serum hTERT levels showed a significant correlation with clinical stage, while correlation with nodal status and tumor size were marginal and no correlation was found with family history and age.ConclusionSerum hTERT is useful for diagnosing and assessing the clinical stage of breast cancer and is superior to conventional markers. Therefore, serum hTERT could have a potential application as a novel biomarker for breast cancer diagnosis.

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18 – C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.