Open Access Original Research Article

Bromate Causes Significant Cytotoxicity and Modulation of the Inflammatory Response

Tebekeme Okoko

Asian Journal of Biotechnology and Genetic Engineering, Page 1-8

Bromate is used as an additive in bread and also a disinfection by-product of hypochlorite treated and ozonated waters. The effect of bromate (as potassium bromate) on the proliferation of U937 cells and activation of U937-derived macrophages was investigated. First, U937 cells were incubated with bromate at different concentrations and later cell viability was assessed via the trypan blue dye staining and reduction of MTT. The production of reactive oxygen species (ROS) was also measured via the DCHF-DA assay. In further experiments, the U937 cells were transformed to the macrophage form via treatment with phorbol-12-myristate-13-acetate before incubated with different concentrations of bromate. Later the production of NO, TNF-a, and IL-6 was evaluated as indices for the activation of macrophages. The results revealed that bromate caused significant cell death when compared to control and was concentration-dependent (p < 0.05). Bromate also significantly produced reactive oxygen species in U937 cells, and also stimulated the production of NO, TNF-a, and IL-6 in U937-derived macrophages. This indicate that bromate causes significant cytotoxicity and also trigger cells to produce significant amount of intracellular reactive oxygen species. Bromate also activated macrophages thus indicating that the cytotoxicity of bromate could involve the modulation of the inflammatory response.

Open Access Original Research Article

Effect of Gibberellic Acid on Seed Germination and Evaluation of Agrobacterium rhizogenes Strains for the Induction of "Hairy Roots" of Phyllanthus niruri Linn. (Phyllantaceae)

Florent Biduaya Mukeba, Gédéon Ngiala Bongo, Johnny Bopopi Mukoko, Freddy Otono Bulubulu, Myriam Mukadi Ngondo, Paulin Kapepula Mutwale, Odette Ngandu Kabena, Koto-te-Nyiwa Ngbolua

Asian Journal of Biotechnology and Genetic Engineering, Page 9-23

Phyllanthus niruri L. is a medicinal plant from tropical regions with great pharmacognosic virtues scientifically proven. It is a rare herbaceous wild species in the dry season. Its use for the pharmaceutical industry requires a regular supply and it is independent of climatic hazards. The main aim to find optimal seed storage conditions with reference to the factors that limit the germination of P. niruri seeds and to promote the emergence of dormancy and germination of P. niruri seeds harvested from different agro-ecological sites in Kinshasa. The results of the present study showed high germination rates with GA3, which significantly influenced the germination of the seeds of Phyllanthus niruri. Seeds stored at laboratory temperature (28°C) had a good germination power than those stored at 4°C. In addition, the germination capacity of seeds stored for 7 days seems to be lower than that of those stored for 14 days. Seeds harvested at low altitude (Kisenso site) gave a higher germination rate than those harvested at high altitude (UPN and CREN-K sites).  However, the late two sites gave almost the same germination rates.

The Agrobacterium rhizogenes strains A4RSpHKN29, LBA 9402 and ATCC 15834 preserved in the Biotechnology Laboratory of CREN-K did not induce the "Hairy roots" of P. niruri after 27 days of culture on MS medium with sucrose but they conserved their plasmids. Nevertheless, some results obtained showed a low production of vitro-plants and transgenic roots (Hairy roots) from caulinary explants.

Open Access Original Research Article

Effects of Ficus sycomorus Phenolic Extracts on the Activity of Hyaluronidase and Phospholipase A2 Enzymes of Echis ocellatus Venom

A. U. Hassan, M. S. Makeri, U. O. Ozojiofor, A. J. Alhassan

Asian Journal of Biotechnology and Genetic Engineering, Page 24-33

Envenomation by snake is one of the most neglected public health problems in Nigeria, causing around 100,000 human deaths annually. It however remains a serious medical, economic and social problem. This research is aimed at evaluating the effects of F. sycomorus extract fractions against Hyaluronidase and Phospholipase A2 (PLA2) enzymes of E. ocellatus snake venom. The plant leaf and stem bark were extracted using chloroform and fractionated using tin Layer and column Chromatography, where flavonoids and tannins were partially purified. Phytochemical analysis of the extracts revealed the presence of tannins, saponin, alkaloids, flavonoids, amino acids, phenols, triterpenoids, terpenoids, steroids and phlobatanins. Gas Chromatography – Mass Spectrometry (GC-MS) analysis done indicates the presence of some basic phenolic compounds, such as; Cyclohexane-1-3,5-trione and 2-phenyl-1,4-benzopyrone. Isolation of the E. ocellatus snake venom enzymes is done using two step processes which include gel filtration on Sephadex G-75 and active fractions were applied to ion–exchange chromatography on Diethylaminoethyl (DEAE) cellulose. The active fractions were then subjected to Soduin Dodecyl Sulphate–Polycrylamide (SDS-PAGE) for molecular weight determination. The partially purified Hyaluronidase and PLA2 gave a total enzyme activity of 1.126904 Tru/Mg and 3.692308 µmol/min and an estimated extrapolated molecular weight of 18 and 14 KDa respectively. IC50 determination of the partially purified plant parts flavonoids and tannins against PLA2 and hyaluronidase from E. ocellatus venom shows the leaf flavonoid extract has the lowest IC50 (Which signifies a higher inhibitory potency) concentration of 0.85 mg/ml and 1.7 mg/ml for hyaluronidase and PLA2 respectively, than all the other fractions tested against both enzymes, while the leaf tannin extract has the highest IC50 (Which signifies a lower inhibitory potency) concentration of 1.65 mg/ml and 2.00 mg/ml. Enzyme inhibition assay carried out reveals the various kinetics parameters for the enzymes in the presence and absence of the inhibitors. Only two of the eight inhibitions, carried out on the partially purified phenolics against the E. ocellatus hyaluronidase and PLA2 enzymes shows a non-competitive kind of inhibition, which is a more promising kind of inhibition and due often turns to an irreversible reaction. F. sycomorus phenolic extracts could provide an alternative natural remedy for the management and treatment of snakebite.

Open Access Original Research Article

Association of Intron 4a/b VNTR NOS3 Gene Polymorphism with Arterial Distensibility in Russian Population

Vladimir N. Melnikov, Sergey G. Krivoschekov, Tamara G. Komlyagina, Irina V. Karmakulova

Asian Journal of Biotechnology and Genetic Engineering, Page 34-41

Nitric oxide is one of the important modulators of arterial tone. It is still unclear whether arterial stiffness depends upon NOS3 gene polymorphism in intron 4. The aim of this observational study was to test an association of central hemodynamic and arterial distensibility with VNTR 4b/a NOS3 gene polymorphism determined by PCR, in Caucasian population.

The routinely ascertained tonometry data were studied by pulse wave analysis using SphygmoCor device in 60 healthy Russian miners (27 women) aged 27–65 years and residing in middle Kola Peninsula (68 degrees N).

Paired comparisons showed that male BB homozygotes had lower values of augmentation pressure (p=.005), and higher brachial-to-aortic pulse pressure amplification (p=.002) than A allele carriers (AB and AA genotypes). These associations remain significant after adjusting for age, heart rate, and systolic blood pressure in multiple regression analysis. Female AB carriers had higher aortic systolic blood pressure (p=.046) and lower subendocardial viability ratio (p=.049) compared to homozygous BB subsample.

Individuals carrying A allele thus have stiffer conduit arteries and seem to be at higher risk for cardiovascular diseases.

Open Access Original Research Article

Genetic Diversity among Ancient Rainfed Lebanese Barley (Hordeum vulgare L.) Landrace Baladi by Using Morphological Traits and Microsatellites Markers

Ali Chehade, Georges Raad, Ahmad El Bitar, Aline Kadri

Asian Journal of Biotechnology and Genetic Engineering, Page 42-50

Lebanese barley landrace Baladi is not sufficiently analyzed morphologically and genetically. This investigation was carried out to evaluate Baladi in its center of diversification by using morphological traits and microsatellite markers (SSR). The genetic diversity of 237 accessions of Lebanese barley landrace Baladi was evaluated by using 19 morphological traits. Principal components analysis showed that plant height, awn color, number of rows, length of rachilla hairs, number of spikelets per spike, number of seeds per spike and shape of lemma tip for sterile spikelets were the most significant traits. Regarding the relationship between plants, dendrogram constructed according to Jaccard distance, clearly separated the studied spikelets into 19 distinguished morphological profiles. A total of 55 genomic DNA extracted from selected accessions were analyzed using seven microsatellite markers (SSRs). A high level of polymorphism information content (PIC; average = 0.66) was observed. The polymorphic primers revealed 16 alleles with an average of 5.3 alleles per polymorphic primer. The highest number of polymorphic bands was developed by the primer pairs MGB402 (7 alleles). In the dendrogram constructed based on the SSR data, the evaluated lines were classified into two major clusters corresponding to the number of rows per spike (two-rows or six-rows). Based on this study, it can be concluded that there is a high level of genetic diversity within Baladi barley landrace in Lebanon. These results will be useful for barley germplasm management in terms of biodiversity protection and as a valuable source of gene pool for new useful alleles for crop improvement.