Expression line approach to recombinant human epidermal growth factor into the yeast,Pichia pastoris from Huh-7 cell line

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.     

Expression Analysis of Fatty Acid Biosynthetic Pathway Genes during Interactions of Oil Palm (Elaeis guineensis Jacq.) with the Pathogenic Ganoderma boninense and Symbiotic Trichoderma harzianum Fungal Organisms

The fatty acid (FA) signaling pathway is emerging as an important mechanism in plant responses during interactions with microbial organisms. For a comprehensive evaluation of key FA biosynthetic pathway genes during interactions of oil palm (Elaeis guineensis Jacq.) with the pathogenic Ganoderma boninense and symbiotic Trichoderma harzianum fungal organisms, a lane-based array analysis of gene expression in artificially inoculated oil palm seedlings was performed. The results obtained demonstrated that acetyl-CoA carboxylase (ACC), β-ketoacyl-ACP synthases (KAS) II and III, palmitoyl-ACP thioesterase (PTE), oleoyl-ACP thioesterase (OTE) and glycerol-3-phosphate acyltransferase (ACT) showed identical responses in root and leaf tissues for the same fungi. The expression of these genes was up-regulated in both root and leaf tissues at 21 days post-inoculation (dpi) during interaction of oil palm with G. boninense. Thereafter, production of physical symptoms occurred at 42 and 63 dpi concomitantly with suppression of expression of these genes. An increase in the expression level of these genes was observed in both tissues at 3–63 dpi, which correlated with the colonization of roots and promotion of plant growth by T. harzianum. These data suggest that FA biosynthetic pathway genes are involved in the defense response of oil palm to infection. Identical plant responses by FA biosynthetic pathway genes may lead to enhanced resistance against G. boninense and could be a useful marker to contribute towards early detection of infection. The distinct expression profile during symbiotic interaction demonstrated its role in plant resistance mechanisms and growth promotion by T. harzianum.     

Cellular impact of combinations of endosulfan,atrazine, and chlorpyrifos on human primary hepatocytes and HepaRG cells after short and chronic exposures

Chronic exposure to low doses of pesticides present in the environment is increasingly suspected to cause major health issues to humans. Toxicological evaluations become more complex when the exposure concerns chemical combinations. Atrazine, chlorpyrifos, and endosulfan are pesticides used worldwide in agriculture and are therefore currently found at residual levels in food and the environment, even in countries in which they are now banned. Our study aimed to use Real-Time Cell Impedance Analyzer to investigate changes in phenotypical status of primary human hepatocytes and differentiated HepaRG cells induced by short and chronic exposures to these three chemicals. In contrast to the traditionally used endpoint cytotoxicity test, this technology allows kinetic measurements in real-time throughout the entire experiment. Our data show significantly higher cytotoxic effects of mixtures as compared to individual pesticides and a greater susceptibility of human hepatocytes as compared to HepaRG to short-term exposure (24 h). Repeated exposure over 2 weeks to endosulfan and endosulfan-containing mixture induced HepaRG cell death in a time- and dose-dependent manner. Of the typical genes involved in metabolism and cell-response to xenobiotics, we found an exposure time- and condition-dependent deregulation of the expression of CYP3A4 and UGT1A in HepaRG cells exposed to low doses of pesticides and mixtures. Our data demonstrate the usefulness of real-time cell monitoring in long-term toxicological evaluations of co-exposure to xenobiotics. In addition, they support but at the same time highlight certain limitations in the use of HepaRG cells as the gold standard liver cell model in toxicity studies.     

Apolipoprotein E genotyping in women with recurrent pregnancy loss: An in silico and experimental hybrid study

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains controversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apolipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of these nonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. The subjects of our current study consisted of 100 women that have had two or more consecutive idiopathic first trimester miscarriages, and one hundred healthy women from the same geographical areas were used as a control group. After DNA extraction, we used a polymerase chain reaction–restriction fragment length polymorphism to genotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutions at codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Phenotyping online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency of the ε4 allele in the case group were statistically and significantly higher than those in the control group (P < 0.05). Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predicted that the amino acid substitution at residue 112 (Apo ε4 allele) is a benign mutation. Accordingly, further studies are required to elucidate the mechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.     

Genetic structure of Chrysanthemum genotypes from Iran assessed by AFLP markers and phenotypic traits

Chrysanthemum is popular worldwide as cut flower, potted plant and perennial garden plant. This study used amplified fragment length polymorphism and phenotypic traits to assess genetic diversity and population structure in Chrysanthemum. Results for the phenotypic traits of ray floret number, tubular floret number, flower number, pedicel length, leaflet number on pedicel and days to visible flower bud had high level (i.e., >30 %) evaluations for genotypic coefficient of variation and phenotypic coefficient of variation; thus, heritability for various phenotypic traits ranged from 45.30 to 94.55 %. Phenotypic data clustered the genotypes into four separate groups. Twenty-five primer combinations were used for molecular analysis. On average, each primer combination produced 83.96 polymorphic DNA bands, ranging from 33 to 122 bands. Polymorphic percentage (99.3 %), polymorphism information content (0.43) and Shannon’s information index (0.45) evaluated high levels of genetic variation in the tested genotypes. The Neighbor-Joining (NJ) method grouped genotypes into six clusters, which were in part confirmed by principal coordinate analysis. A Bayesian structure analysis identified four clusters, in which 30 individuals were maintained within the admixed clusters. Results from this study provide appropriate information applicable to designing effective breeding programs and other analyses associated with future studies of Chrysanthemum.     

Plasmon analysis in wheat alloplasmic lines using morphological and chloroplast microsatellite markers

Inheritance of plant traits mainly depends upon nuclear genes, cytoplasmic factors and their interactions. In the present study, 32 alloplasmic lines accompanied by a euplasmic parental line (control) were evaluated using molecular (chloroplast microsatellite) and morpho-physiological traits during 2010–2011 and 2011–2012. The results of combined analysis of variance showed the significant effect of growing seasons on most of the studied traits as well as the significant effect of cytoplasm on plant height, leaf net CO₂ assimilation rate and grain yield per plant. Results of cluster analysis divided the six plasmons based on their phenotypic effects into three groups: (1) R and S^v type, D (Aegilops typica and Ae. ventricosa) and D² type, as well as B-type plasmon (euplasmic line); (2) a single plasmon of M type and three plasmons of B type; (3) all other B-type and a single D-type plasmon (Aegilops cylindrica). Molecular analysis showed that 20 primer pairs out of 26 chloroplastic microsatellite markers (cpSSR) produced polymorphic bands in the alloplasmic lines. A total of 50 alleles were identified with an average of 2.5 alleles per locus. In this study, polymorphism information content (PIC) ranged from 0.05 (WCt17 primer) to 0.49 (WCt9 primer). Cluster analysis of molecular data revealed that the alloplasmic lines belong to two major clusters, in which differentiation of cytoplasmic types belonging to the genus Triticum and Aegilops has been evident. Likewise, analysis of molecular variance showed significant differences between two studied groups (F _ST = 0.67, P < 0.001). Overall, our findings indicated that the cpSSR markers can be valuable resources of polymorphic markers for the analysis of cytoplasm of Triticeae species, with the potential for clear differentiation in close species and genera of this tribe.     

Assessment of probiotic potential and anticancer activity of newly isolated vaginal bacterium Lactobacillus plantarum 5BL

Numerous bacteria in and on its external parts protect the human body from harmful threats. This study aimed to investigate the potential beneficial effects of the vaginal ecosystem microbiota. A type of bacteria was isolated from vaginal secretions of adolescent and young adult women, cultured on an appropriate specific culture medium, and then molecularly identified through 16S rDNA gene sequencing. Results of 16S rDNA sequencing revealed that the isolate belongs to the Lactobacillus plantarum species. The isolated strain exhibited probiotic properties such as low pH and high bile salt concentration tolerance, antibiotic susceptibility and antimicrobial activity against some pathogenic bacteria. The anticancer effects of the strain on human cancer cell lines (cervical, HeLa; gastric, AGS; colon, HT‐29; breast, MCF‐7) and on a human normal cell line (human umbilical vein endothelial cells [HUVEC]) were investigated. Toxic side effects were assessed by studying apoptosis in the treated cells. The strain exhibited desirable probiotic properties and remarkable anticancer activity against the tested human cancer cell lines (P ≤ 0.05) with no significant cytotoxic effects on HUVEC normal cells (P ≤ 0.05). Overall, the isolated strain showed favorable potential as a bioactive therapeutic agent. Therefore, this strain should be subjected to the other required tests to prove its suitability for clinical therapeutic application.     

Investigations of anticholinestrase and antioxidant potentials of methanolic extract,subsequent fractions,crude saponins and flavonoids isolated from Isodon rugosus

Background

Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer’s and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman’s spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H₂O₂ and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively.

Results

Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC₅₀ values of 45, 50 for AChE and 40, 70 μg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC₅₀ values of 20 and 47 μg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H₂O₂ assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC₅₀ of 19 and 24 μg/ml against DPPH and H₂O₂ radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities.

Conclusion

It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.     

Phytochemical and toxicological investigations of crude methanolic extracts,subsequent fractions and crude saponins of Isodon rugosus

Background

Isodon rugosus is used traditionally in the management of hypertension, rheumatism, tooth-ache and pyrexia. Present study was arranged to investigate I. rugosus for phytoconstituents, phytotoxic and cytotoxic activities to explore its toxicological, pharmacological potentials and to rationalize its ethnomedicinal uses. Briefly, qualitative phytochemical analysis of the plant extracts were carried out for the existence of alkaloids, flavonoids, saponins, oils, glycosides, anthraquinones, terpenoids, sterols and tannins. Plant crude methanolic extract (Ir.Cr), its subsequent fractions; n-hexane (Ir.Hex), chloroform (Ir.Chf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq) and saponins (Ir.Sp) in different concentrations were tested for phytotoxic and cytotoxic activities using radish seeds and brine shrimps (Artemia salina) respectively. The phytotoxic activity was determined by percent root length inhibition (RLI) and percent seeds germination inhibition (SGI) while the cytotoxicity was obtained with percent lethality of the brine shrimps.

Results

Ir.Cr was tested positive for the presence of alkaloids, glycosides, flavonoids, oils, terpenoids, saponins, tannins and anthraquinones. Among different fractions Ir.Sp, Ir.Chf, Ir.EtAc, and Ir.Cr were most effective causing 93.55, 89.32, 81.32 and 58.68% inhibition of seeds in phytotoxicity assay, with IC₅₀ values of 0.1, 0.1, 0.1 and 52 μg/ml respectively. Similarly, among all the tested samples, Ir.Sp exhibited the highest phytotoxic effect causing 91.33% root length inhibition with IC₅₀ of 0.1 μg/ml. Ir.Sp and Ir.Chf were most effective against brine shrimps showing 92.23 and 76.67% lethality with LC₅₀ values of 10 and 12 μg/ml respectively.

Conclusions

It may be inferred from the current investigations that I. rugosus contains different secondary metabolites and is a potential source for the isolation of natural anticancer and herbicidal drug molecules. Different fractions exhibited phytotoxic and cytotoxic activities, thus providing pharmacological basis for ethnomedicinal uses of this plant.