Joseph Madassery

RAPD ANALYSIS OF A VARIANT OF BANANA (MUSA SP.) CV. GRANDE NAINE AND ITS PROPAGATION VIA SHOOT TIP CULTURE

SummaryA morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N6-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was multiplied using shoot tip culture, and the shoots were rooted on half-strength MS medium supplemented with 2.69 μM α-naphthaleneacetic acid. All plantlets showed variegated leaves under field conditions.

Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-α.

Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.

RAPID IN VITRO PRODUCTION OF TRUE-TO-TYPE PLANTS OF POGOSTEMON HEYNEANUS THROUGH DEDIFFERENTIATED AXILLARY BUDS

SummaryRapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N6-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitro-derived plantlets. At the optimum level of BA (2.22μM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40d. Retaining the culture without subenlture enhanced the number of shoots (>30 shoots). Inereased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60d) initiated 25–30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22μM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40d. Subeulture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 μM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristies identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns.

DIRECT AND INDIRECT SOMATIC EMBRYOGENESIS ON COTYLEDON EXPLANTS OF QUASSIA AMARA L., AN ANTILEUKAEMIC DRUG PLANT

SummaryIn vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N6-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.

High-Frequency Transgenic Plant Regeneration and Plumbagin Production through Methyl Jasmonate Elicitation from Hairy Roots of Plumbago indica L.

High-frequency transgenic plant regeneration and production of plumbagin were accomplished from hairy roots induced by Agrobacterium rhizogenes strain A4M70GUS on Plumbago indica L. Of the two types of hairy roots developed on Murashige and Skoog (MS) basal medium, Type I was long and thick with lesser branches and root hairs, and Type II was highly-branched short and slender with tufts of root hairs. Of the different lines grown in half-strength MS liquid basal media, root line 5 (R5) of the Type I yielded the highest plumbagin (0.92% DW) and was significantly different to that of the in vitro control. R5 showed a stable production of plumbagin (1.09% DW) in subsequent cultures. Elicitation of R5 with 50 μM methyl jasmonate for 48 h increased the yield of plumbagin to 5.0% DW, and was superior to 100 μM acetylsalicylic acid (3.8% DW). Plumbagin yield was five times that of twoyear-old ex vitro roots. Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the transformation. Hairy root segments cultured on MS medium containing 8.8 μM benzyladenine and 2.5 μM indole-3-butyric acid induced a mean of 9.1 shoots. Subsequent culture of the isolated shoots developed more than 50 normal shoots per culture. The root-free shoots were rooted on half-strength MS basal medium. The plantlets transferred in field conditions grew normally and exhibited 90% survival. Transgenic plant regeneration and hairy root induction in P. indica serves as reliable source of plumbagin which in turn cut off the mass destruction of the plant species.

Genetic Diversity Assessment of Rarely Cultivated Traditional Indica Rice (Oryza sativa L.) Varieties

Random amplified polymorphic DNA fingerprinting was performed to assess the genetic diversity among rarely cultivated traditional indica rice (Oryza sativa L.) varieties collected from a tribal hamlet of Kerala State, India. A total of 664 DNA bands amplified by 15 primers exhibited 72.9% polymorphism (an average of 32.3 polymorphic bands per primer). The varieties Jeerakasala and Kalladiyaran exhibited the highest percent (50.19%) polymorphism, while Thondi and Adukkan showed the lowest (9.85%). Adukkan (78 bands) and Jeerakasala (56 bands) yielded the highest and the lowest number of amplicons, respectively. Unweighted Pair Group Method with Arithmetic mean analysis using the Dice similarity coefficient showed the highest value of similarity coefficient between the varieties Adukkan and Thondi, both shared higher level of similarity (0.81), followed by Kanali and Thondi (0.88). Of the three subclusters, the varieties of Adukkan, Thondi, Kanali, Mannuveliyan, Thonnuranthondi, and Chennellu grouped together with a similarity of 0.77. The second group represented by Navara, Gandhakasala, and Jeerakasala with a similarity coefficient of 0.76 formed a cohesive group. The variety Kalladiyaran formed an isolated position that joined the second cluster. The Principal Coordinate Analysis also showed separation of Kalladiyaran from the other varieties.

Tetracycline and Glutathione Inhibit Matrix Metalloproteinase Activity: An In Vitro Study Using Culture Supernatants of L929 and Dalton Lymphoma Cell Lines

Tetracycline and glutathione inhibited the protease activities of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressed by mouse fibrosarcoma cells (L929) and Dalton lymphoma cells, respectively. The inhibitory activity of the tetracycline may be due to its ability to chelate metal ions such as calcium and zinc. Gelatin-zymography technique was used to demonstrate the inhibitory activity of both tetracycline and glutathione. The intensity of the bands corresponding to metalloproteinase activity in zymography gel was reduced in the presence of 50–100 μg/mL of tetracycline. The presence of 10–100 μg/mL of tetracycline in the medium increased the adherence of L929 cancer cells. These results clearly indicate the antimetastatic property of tetracycline. Reduced glutathione, a compound which is produced endogenously by the cells to maintain the redox status, was shown to inhibit the matrix metalloproteinase activity (in vitro). Therefore, it is assumed that decreased glutathione levels in synovial fluids or plasma might increase the activity of MMP. Reduced glutathione at 100 μg/mL inhibited the metalloproteinase activity in gelatin-zymographic gel. As both tetracycline and glutathione exhibited an inhibitory effect on matrix metalloproteinase activity, it was of great interest to check their clinical effects on various MMP associated pathological conditions such as cancer metastasis and arthritis. Here we report that tetracycline and reduced glutathione inhibited the activity of MMP2 completely and activity of MMP9 partly.

Cytotoxic Effect of (Z)-Ethylidene-4,6-Dimethoxycoumaran-3-One Isolated from Pogostemon quadrifolius (Benth.) on PC-3 and DU-145 Prostate Cancer Cells

The study was conducted to identify the antiproliferative property and the mode of action of (Z)-ethylidene-4,6-dimethoxycoumaran-3-one (EDC), a novel compound isolated from the plant leaves of Pogostemon quadrifolius (Benth.), against prostate cancer cell lines PC-3 and DU-145. EDC inhibited the proliferation of PC-3 and DU-145 cell lines at micro molar concentrations. EDC strongly inhibited the clonogenic propagation of prostate cancer cells. EDC arrests the prostate cancer cells at G2/M phase of the cell cycle and then eventually leads to apoptosis, which was confirmed by DNA fragmentation assay and poly (ADP-ribose) polymerase cleavage western blot analysis. At sub-lethal concentrations, EDC inhibited the migration of PC-3 by 87.1% and DU-145 by 68.0%. Thus, the present study revealed the in vitro cytotoxic and anti-metastatic property of EDC against PC-3 and DU-145 cancer cells.

Andrographolide, a new potential NF-κB inhibitor: docking simulation and evaluation of drug-likeness

Andrographolide is a potent anticancer and anti-inflammatory agent isolated from the plant Andrographis paniculata. It is found to be cytotoxic against various cancer cell lines (in vitro) and also reported to act as an anti-inflammatory agent by interfering with NF-κB protein. Andrographolide induced higher percentage of apoptosis in glutathione-depleted lymphoma cell lines. Andrographolide was also reported to form dehydrated adduct with reduced glutathione at 50° C. On the basis of these observations, the docking analysis of andrographolide with its target protein (NF-κB/p50) and its proposed anti-target protein (glutathione S-transferase) was carried out. Docking analysis predicted the best putative pose of andrographolide molecule in the active site of NF-κB and glutathione S-transferase proteins. Here we report that the furan ring of andrographolide interacts with cysteine 59 of NF-κB/p50 and thereby inhibiting the protein action. Docking studies showed the andrographolide binding to the H-site of glutathione S-transferase enzyme which resembles the behaviour of susceptible xenobiotics inactivated by glutathione S-transferase enzyme. Andrographolide obeys Pfizers rule but drug-likeness value for andrographolide is found to be negative as the molecule showed low drug score. Hence, inactivation by glutathione S-transferase and low drug score could possibly be the reasons to make andrographolide inactive in clinical trial.

Bioassay-guided isolation and evaluation of antiproliferative effects of (Z)-ethylidene-4, 6-dimethoxycoumaran-3-one from Pogostemon Quadrifolius (Benth.)

The aim of the study was to isolate and identify the major cytotoxic principle from plant leaves of Pogostemon quadrifolius (Benth.) and evaluate its antiproliferative potential against human cancer cells. Plant leaves were extracted sequentially with a soxhlet apparatus, using petroleum ether, chloroform and methanol solvents. Petroleum ether and chloroform extracts exhibited antiproliferative properties against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1cancer cell lines tested, but methanol extracts failed to exhibit such activity. The major antiproliferative principle from petroleum ether and chloroform extracts was isolated with the help of bioassay guided column chromatography. This cytotoxic compound was further analysed by UV, TLC, HPLC, LC-MS, GC-MS and NMR analyses and was identified to be novel: (Z)-ethylidene-4,6-dimethoxycoumaran-3-one (Compound 1). The half-maximal inhibitory concentrations for proliferation (IC50) exhibited by compound 1 were 19.4, 23.1, 22.1, 35.9 and 8.32 µM against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1 cancer cell lines, respectively. Further experiments revealed that compound 1 triggered the apoptosis mode of cell death in cancer cell lines. Thus, the present study allowed isolation and identification of a novel cytotoxic natural compound, (Z)-ethylidene-4,6-dimethoxycoumaran-3-one, from plant leaves of P. quadrifolius (Benth.). Our pre-clinical study also indicated that compound 1 is particularly active in the acute T cell leukemia cell line (Jurkat E6-1) with potential for application as a chemotherapeutic agent in the future.