In vitro cytogenetic studies of cypermethrin on human lymphocytes

Assessment of cytotoxicity and response to external factors like pesticides were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) or MTT assay, which measures mitochondrial metabolism in the entire cell culture and provides information about the percentage of cell survival. Utilizing the MTT assay, the cytotoxicity of cypermethrin was determined on lymphocyte cultures from human peripheral blood samples, the short-term lymphocyte cultures were incubated with various aliquots of the cypermethrin and the LC50 was found to be 33.6 microM. Lymphocytes treated with low-doses (1/10 of LC50) of cypermethrin showed an increase in the frequency of chromosomal aberrations and found to be significant. Karyotype analysis revealed more satellite associations and chromosomal breaks in cypermethrin treated samples. Low-doses of the pesticide also induced single-strand breaks in the DNA as assessed by comet assay. The pesticide caused increase in the comet tail length with increase in pesticide concentration, implicating genotoxicity in somatic cells. It is concluded that In vitro assays could give important information of the mechanism of toxicity at low dosages and impact on genetic material of human origin.     

Taxoid from the needles of the Himalayan yew Taxus wallichiana with cytotoxic and immunomodulatory activities

The needles of Taxus wallichiana gave a taxoid 1-hydroxy- 2-deacetoxy-5-decinnamoyl-taxinine j, whose structure has been established by spectroscopic data and confirmed by X-ray crystallography. The taxoid possesses significant cytotoxic and immunomodulatory activity.     

Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant

Cannabis sativa L. (Cannabaceae) is an important medicinal plant well known for its pharmacologic and therapeutic potency. Because of allogamous nature of this species, it is difficult to maintain its potency and efficacy if grown from the seeds. Therefore, chemical profile-based screening, selection of high yielding elite clones and their propagation using biotechnological tools is the most suitable way to maintain their genetic lines. In this regard, we report a simple and efficient method for the in vitro propagation of a screened and selected high yielding drug type variety of Cannabis sativa, MX-1 using synthetic seed technology. Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 % sodium alginate with 50 mM CaCl₂.2H₂O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 %) under in vitro conditions. Under in vivo conditions, 100 % conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose and 0.5 % PPM. Plantlets regenerated from the encapsulated explants were hardened off and successfully transferred to the soil. These plants are selected to be used in mass cultivation for the production of biomass as a starting material for the isolation of THC as a bulk active pharmaceutical.Key words: Encapsulation, Nodal explants, Plant growth regulators, Plant regeneration, Synthetic seeds     

Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.     

Variation in morphology and morphometrics of eggs of Culex quinquefasciatus mosquitoes from different ecological regions of India

Variation in egg surface morphology and morphometrics of Culex quinquefasciatus mosquitoes of the Jodhpur, Bikaner, Jamnagar, and Bathinda strains were correlated with geographical distribution in different ecological regions of India. We report the geographic variation in Cx. quinquefasciatus based on 44 attributes of micropylar and conical‐shaped regions of eggs, including micropylar apparatus (corolla, disc, and mound), micropylar tubercles, and the exochorionic tubercle, pores, and network in anterior, middle, and posterior regions. No remarkable differences were observed in the surface morphology of eggs of these strains except the absence of small tubercles in the anterior and middle region of the JMN strain. However, a statistical analysis indicated significant morphometric variations in 66% of the attributes of the eggs. The cluster analysis of all egg attributes showed that the JD, BKN, and BTH strains are closer to each other than the JMN strain. The positive correlation (r = 0.95) also indicated an effect of geographical distribution on morphometry of various egg attributes of these strains. The present study suggests that ecological variation may have affected the morphometric attributes of the egg of four strains of Cx. quinquefasciatus from different geographical areas.     

Synthesis of 2,4,6-trisubstituted pyrimidine and triazine heterocycles as antileishmanial agents

A series of 2,4,6 trisubstituted pyrimidines and triazines have been synthesized and screened for its in vitro antileishmanial activity profile in promastigote model. Nine compounds have shown > 94% inhibition against promastigotes at a concentration of 10 microg/mL.     

Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.Lactic acid (lactate) and its derivatives have many applications in the food, pharmaceutical, and polymer industries (13, 30). An example is polylactic acid, a renewable, biodegradable, and environmentally friendly polymer produced from d- and l-lactate (19). In this context, biological processes have the advantage of being able to produce chirally pure lactate from inexpensive media containing only the carbon source and mineral salts (43). While lactic acid bacteria traditionally have been used in the production of d-lactate from carbohydrate-rich feedstocks, several laboratories recently have reported alternative biocatalysts (13, 30), many of which are engineered Escherichia coli strains that produce d- or l-lactate (4, 8, 50, 51, 52).Unlike the aforementioned reports, which have dealt with the use of carbohydrates, our work focuses on the use of glycerol as a carbon source for the production of d-lactate. Glycerol has become an inexpensive and abundant substrate due to its generation in large amounts as a by-product of biodiesel and bioethanol production (18, 32, 47). The conversion of glycerol to higher-value products has been proposed as a path to economic viability for the biofuels industry (47). One such product is lactate, whose production could be readily integrated into existing biodiesel and bioethanol facilities, thus establishing true biorefineries.Although many microorganisms are able to metabolize glycerol (25), the use of industrial microbes such as E. coli could greatly accelerate the development of platforms to produce fuels and chemicals from this carbon source. We recently reported on the ability of E. coli to metabolize glycerol under either anaerobic or microaerobic conditions and identified the environmental and metabolic determinants of these processes (9, 11, 28). In one of the studies, the pathways involved in the microaerobic utilization of glycerol were elucidated, and they are shown in Fig. ​Fig.11 (9). A common characteristic of glycerol metabolism under either anaerobic or microaerobic conditions is the generation of ethanol as the primary product and the negligible production of lactate (6, 9, 11, 28). In the work reported here, the knowledge base created by the aforementioned studies was used to engineer E. coli for the efficient conversion of glycerol to d-lactate in minimal medium. The engineered strains hold great promise as potential biocatalysts for the conversion of low-value glycerol streams to a higher-value product like d-lactate.Open in a separate windowFIG. 1.Pathways involved in the microaerobic utilization of glycerol in E. coli (9). Genetic modifications supporting the metabolic engineering strategies employed in this work are illustrated by thicker lines (overexpression of gldA-dhaKLM, glpK-glpD, and ldhA) or cross bars (disruption of pflB, pta, adhE, frdA, and dld). Broken lines illustrate multiple steps. Relevant reactions are represented by the names of the gene(s) coding for the enzymes: aceEF-lpdA, pyruvate dehydrogenase complex; adhE, acetaldehyde/alcohol dehydrogenase; ackA, acetate kinase; dhaKLM, dihydroxyacetone kinase; dld, respiratory d-lactate dehydrogenase; fdhF, formate dehydrogenase, part of the formate hydrogenlyase complex; frdABCD, fumarate reductase; gldA, glycerol dehydrogenase; glpD, aerobic glycerol-3-phosphate dehydrogenase; glpK, glycerol kinase; hycB-I, hydrogenase 3, part of the formate hydrogenlyase complex; ldhA, fermentative d-lactate dehydrogenase; pflB, pyruvate formate-lyase; pta, phosphate acetyltransferase; pykF, pyruvate kinase. Abbreviations: DHA, dihydroxyacetone; DHAP, DHA phosphate; G-3-P, glycerol-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; P/O, amount of ATP produced in the oxidative phosphorylation per pair of electrons transferred through the electron transport system; QH₂, reduced quinones.     

A semianalytical model to study the effect of cortical tension on cell rolling

Cell rolling on the vascular endothelium plays an important role in trafficking of leukocytes, stem cells, and cancer cells. We describe a semianalytical model of cell rolling that focuses on the microvillus as the unit of cell-substrate interaction and integrates microvillus mechanics, receptor clustering, force-dependent receptor-ligand kinetics, and cortical tension that enables incorporation of cell body deformation. Using parameters obtained from independent experiments, the model showed excellent agreement with experimental studies of neutrophil rolling on P-selectin and predicted different regimes of cell rolling, including spreading of the cells on the substrate under high shear. The cortical tension affected the cell-surface contact area and influenced the rolling velocity, and modulated the dependence of rolling velocity on microvillus stiffness. Moreover, at the same shear stress, microvilli of cells with higher cortical tension carried a greater load compared to those with lower cortical tension. We also used the model to obtain a scaling dependence of the contact radius and cell rolling velocity under different conditions of shear stress, cortical tension, and ligand density. This model advances theoretical understanding of cell rolling by incorporating cortical tension and microvillus extension into a versatile, semianalytical framework.