R. B. Subramanian

Extraction and purification of phytol from Abutilon indicum: cytotoxic and apoptotic activity

Medicinal plants possess a wide range of secondary metabolites and most of these secondary metabolites have their own importance in the field of medicine. Abutilon indicum is widely used as a medicinal plant in the Indian system of medicine. In the present study, we have evaluated the apoptosis inducing ability of leaf extract of A. indicum. Hydromethanolic leaf extract showed cytotoxicity on Schizosaccharomyces pombe cells by affecting the growth and viability. Phytochemical analysis of the extract revealed the presence of secondary metabolites. A 2D TLC method was used for the separation of the active cytotoxic compound. A HPTLC method was used for the quantification of the bioactive compound. A GC-HRMS method was employed for the identification of the active principle as phytol, which was found to be responsible for inducing ROS mediated apoptosis in S. pombe.

Phytol induces ROS mediated apoptosis by induction of caspase 9 and 3 through activation of TRAIL, FAS and TNF receptors and inhibits tumor progression factor Glucose 6 phosphate dehydrogenase in lung carcinoma cell line (A549)

A number of drugs as well as lead molecules are isolated from natural sources. Phytol is one of such lead molecule belongs to terpenes group distributed widely in medicinal plants. In the present work, we investigated the cytotoxic behavior of phytol on human lung carcinoma cells (A549). Phytol was found to cause characteristic apoptotic morphological changes and generation of ROS in A549 cells. The mechanism of phytol involved the activation of TRAIL, FAS and TNF-α receptors along with caspase 9 and 3. In silico molecular docking studies revealed that phytol has a good binding affinity with glucose-6-phosphate dehydrogenase (G6PD), which is known to promote tumor proliferation. The ability of phytol to become potential drug candidate has been revealed from the pharmacokinetic study performed in the present study.

Optimization of Process Parameters for Cellulase Production by Bacillus licheniformis MTCC 429 using RSM and Molecular Characterization of Cellulase Gene

World is threaten to energy crisis which has advances research in bioenergy and specifically development of biofuels to replace petroleum products have increased the use of microbial enzyme like cellulases and xylanases as well as amylases for generation of reducing sugars for their conversion into bioethanol. Extensive research has been carried out in this view but alkaline cellulase production and molecular characterization is not studied in detail so far, this study will aid to achieve it. Optimization of fermentation parameters for production of cellulase was evaluated with the help of Response Surface Methodology (RSM) a statistical design, initial pH (9), moisture ratio (1:1) and incubation time (72 h) (run no.4) were found to be ideal parameters for optimum production of cellulase, substrate Jatropha seed cake without any pre-treatment was found to be an ideal source for cellulase production by Bacillus licheniformis under solid state fermentation. Cellulase gene of size 786 bp was isolated later using PCR techniques, confirmed with sequence analysis and ligated to pRSET A vector for the transformation to E. coli DH5α. Positive clones were identified and sequenced to justify the cloning. Sequence of Bacillus licheniformis endo-β-1,4-glucanase (Cel12A) gene showed 100% similarity with endoglucanase gene sequence from Bacillus licheniformis ATCC 14580 genome, shows successful cloning of Cel12A gene into pRSET A vector.

Purification and properties of an endoglucanase from Thermoascus aurantiacus

An Endo-cellulase was purified to homogeneity using ammonium sulfate precipitation, ion exchange and size exclusion chromatography from newly isolated strain of Thermoascus aurantiacus RBB-1. The recovery and purification fold were 13.3% and 6.6, respectively, after size exclusion chromatography. The purified cellulase has a molecular mass (M) of 35 kDa. Optimum temperature for the enzyme was found to be 70 °C and stability was upto 80 °C for 1 h. Along with higher stability at 80 °C, enzyme showed half lives of 192 h and 144 h at 50 and 70 °C respectively. The purified cellulase was optimally active at pH 4.0 and was stable over a broad pH range of 3.0–7.0. The enzyme purified showed apparent Km and Vmax values of 37 mg/ml and 82.6 U/min/mg protein respectively with higher salt tolerance of 10% for 1 h.

Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

L-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel L-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7IU/mg, which is higher than native (rBliA) (407.65IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with Km value of 0.42mM and Vmax of 2778.9μmolmin(-1).

Nickel accumulation by Colocassia esculentum and its impact on plant growth and physiology

The remediation of heavy metal-contaminated sites using plants presents a promising alternative to current methodologies. In this study, the potential of Colocassia esculentum for Nickel (Ni) accumulation was determined. C. esculentum plants exposed to Ni, demonstrated capability to accumulate on average, more in shoots as compared to roots, suggesting better translocation of Ni from root to shoot. High metal content in soil caused reduction in growth parameters and an increase in oxidative stress. Under heavy metal stress, an increase in catalase, peroxidase, ascorbic acid and proline were observed in the roots along with some anatomical changes. Lipid peroxidation showed a slight increase by Ni treatment along with some anatomical changes in the root. This work demonstrates that metal induced oxidative stress occurs by the presence of heavy metals at higher concentrations. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in C. esculentum. The outcome of this study corroborate that C. esculentum is a suitable candidate for the phytoremediation of Ni contaminated soil and could be considered as a potential Ni hyperaccumulator plant species.

Isolation of NBS-LRR class resistant gene (I2 gene) from tomato cultivar Heamsona

Three races of Fusarium oxysporum f. sp. lycopersici race 1, 2 and 3 are identified depending on the avirulence protein or effector protein secreted by fungal pathogen during the host colonization in tomato. These effector proteins are recognized by the host innate immune system based on R gene expressions that are I1, I2 and I3 in tomato for each races. Amongst the three, I2 protein has been cloned and characterized for the incompatibility against race 2 type of the pathogens. In India race 1 type of F. oxysporum f. sp. lycopersici observed commonly which require presence of I1 gene in tomato plant for the incompatibility reactions but in the present study, I2 gene was partially isolated from the tomato cultivar Heamsona and observed to be resistance against race 1 type of pathogen. Keywords: Fusarium wilt, race, R-gene, resistance, tomato. African Journal of Biotechnology Vol. 12(42), pp. 6076-6078

Anatomical and biochemical aspects of interaction between roots of chickpea and Fusarium oxysporum f. sp. ciceris race 2

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots.

Single nucleotide variant detection in Jaffrabadi buffalo (Bubalus bubalis) using high-throughput targeted sequencing

The water buffalo is among the most important livestock species of southern Asia, contributing greatly to the ecosystem and rural livelihood of the region. The identification of large-scale single nucleotide polymorphisms in this species would greatly facilitate our understanding of the genetic basis of economically important traits such as milk production, fertility traits and general health traits. The present study investigated the cost-effective method of exome capture and single nucleotide variant (SNV) identification from genomic DNA of Jaffrabadi buffalo using biotin-labelled cDNA as probes. Sequencing of enriched fragments generated 608 Mb of data, which was mapped to a Bos taurus genome assembly followed by variant calling and annotation. Furthermore, 393 coding SNVs were identified, leading to 143 non-synonymous substitutions (nsSNVs) in 75 genes. Of the 75 nsSNV-containing genes, four matched the genes that have previously been reported to be potentially associated with economically important traits such as milk production and meat production. Furthermore, functional annotation using gene ontology (GO) enrichment identified categories such as glutamate receptor activity (GO: 0008066) enriched in the fertility trait samples. These results provide a framework for the application of cost-effective methods of target capture in SNV detection from non-model organisms such as the water buffalo.