Chelliah Jayabaskaran

Biotechnological potential of plant-associated endophytic fungi: hope versus hype.

The potential of endophytes, particularly endophytic fungi, capable of demonstrating desirable functional traits worth exploitation using red biotechnology is well established. However, these discoveries have not yet translated into industrial bioprocesses for commercial production of biopharmaceuticals using fungal endophytes. Here, we define the current challenges in transforming curiosity driven discoveries into industrial scale endophyte biotechnology. The possible practical, feasible, and sustainable strategies that can lead to harnessing fungal endophyte-mediated pharmaceutical products are discussed.

Production of paclitaxel by Fusarium solani isolated from Taxus celebica

A fungus was isolated from the stem cuttings of Taxus celebica, which produced paclitaxel in liquid-grown cultures. The fungus was identified as Fusarium solani based on colony characteristics, morphology of conidia and the 26S rDNA sequence. Paclitaxel was identified by chromatographic and spectroscopic comparison with authentic paclitaxel and its cytotoxic activity towards Jurkat cells in vitro.

Rethinking production of Taxol® (paclitaxel) using endophyte biotechnology

Taxol® (generic name paclitaxel) represents one of the most clinically valuable natural products known to mankind in the recent past. More than two decades have elapsed since the notable discovery of the first Taxol®-producing endophytic fungus, which was followed by a plethora of reports on other endophytes possessing similar biosynthetic potential. However, industrial-scale Taxol® production using fungal endophytes, although seemingly promising, has not seen the light of the day. In this opinion article, we embark on the current state of knowledge on Taxol® biosynthesis focusing on the chemical ecology of its producers, and ask whether it is actually possible to produce Taxol® using endophyte biotechnology. The key problems that have prevented the exploitation of potent endophytic fungi by industrial bioprocesses for sustained production of Taxol® are discussed.

Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation.

Naphthalene Carbohydrazone Based Dizinc(II) Chemosensor for a Pyrophosphate Ion and Its DNA Assessment Application in Polymerase Chain Reaction Products

A new naphthalene carbohydrazone based dizinc(II) complex has been synthesized and investigated to act as a highly selective fluorescence and visual sensor for a pyrophosphate ion with a quite low detection limit of 155 ppb; this has also been used to detect the pyrophosphate ion released from polymerase-chain-reaction.

Distinct light-, cytokinin- and tissue-specific regulation of calcium dependent protein kinase gene expression in cucumber (Cucumis sativus)

Abstract In plants, calcium dependent protein kinases (CDPKs) constitute a unique family of enzymes that is characterized by a C-terminal calmodulin (CaM)-like domain. In this study, we have cloned four partial CDPK cDNAs (CsCDPK1–4) from cucumber by reverse transcription polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CDPKs. Transcript levels of one of the CDPK messengers viz. CsCDPK3 were measured in intact, etiolated, excised cotyledons, hypocotyls and roots following treatments with light or phytohormones (cytokinin/auxin) using a recently evolved high-sensitivity quantitative RT-PCR method (TaqMan analysis). The highest transcript levels of CsCDPK3 were detected in hypocotyls followed by roots and cotyledons. Exposure to light was found to have a down-regulatory effect on CsCDPK3 transcript levels in excised hypocotyls and roots unlike in excised cotyledons where light was found to exert an up-regulatory effect. Treatment with benzyladenine (cytokinin) up-regulated CsCDPK3 transcript levels in cotyledons as opposed to a down-regulatory effect in roots and did not seem to have a significant effect on CsCDPK3 transcript levels in hypocotyls. On the other hand, 2,4-dichlorophenoxyacetic acid (2,4-D) (auxin) treatments did not cause any significant changes in CsCDPK3 transcript levels in hypocotyls, cotyledons or roots. Thus, our results show that light and cytokinin differentially regulate CsCDPK3 transcript levels in a tissue-specific manner.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death.

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp—CrP14, obtained from stem tissues, and Talaromyces radicus—CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 μg/ml and 20 μg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus—CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus—CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 μg/l) in modified M2 medium and of vinblastine (70 μg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

In Vitro Cytotoxicity and Apoptosis Induction in Human Cancer Cells by Culture Extract of an Endophytic Fusarium solani Strain Isolated from Datura metel L

Objectives: Endophytic strains of many plants-particularly medicinal plants-produce a plethora of secondary metabolites, many of which are of immense pharmaceutical importance e.g. anticancer properties. Datura metel L., an important medicinal plant in use as a topical application to remove tumors apart from its other medicinal uses, has not been extensively explored for the potential of its endophytes for their pharmacological significance. The present study was thus initiated to investigate the anticancer effects of the organic extract of an endophytic fungus isolated from this plant. Methods: We report the anticancer effects of an endophytic Fusarium solani fungal strain isolated from Datura metel L. as evaluated by its culture extract on five human cancer cell lines (HepG2, HeLa, MCF-7, OVCAR-3 and PC-3). The ethyl acetate (EtOAc) extract of three week grown fungal culture was tested for its cytotoxic activity on different cancer cell lines by MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The apoptosis-inducing activity and its effect on mitochondrial membrane potential were measured by flow cytometry using JC-1 dye. Nuclear DNA condensation was assessed by fluorescence microscopy using Hoechst 33342 stain. DNA fragmentation was visualized by gel electrophoresis. Results: The ethyl acetate (EtOAc) fungal culture extract showed cytotoxic activity against all the tested human cancer cell lines, in particular against cervical cancer cells HeLa. Further, loss of mitochondrial membrane potential, DNA fragmentation and nuclear chromatin condensation strongly support the ability of organic extract to induce the cancer cell apoptosis though the mitochondrial pathway. Conclusion: The results demonstrate that the Fusarium solani organic extract harbor potential anticancer lead compound(s) which inhibited the proliferation of various cancer cells by inducing cell apoptosis.

Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons : involvement of Ca2+ and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal

Light- and cytokinin-regulated ftsZ gene expression in excised cucumber cotyledons (Cucumis sativus)

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