Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks

Background and Aim: Humic substances (HSs) influence the chemical and physical properties of the soil, and are also known to affect plant physiology and nutrient uptake. This study aimed to elucidate plant metabolic pathways and physiological processes influenced by HS activity. Methods: Arabidopsis roots were treated with HS for 8 h. Quantitative mass spectrometry-based proteomics analysis of root proteins was performed using the iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. Out of 902 protein families identified and quantified for HS treated vs. untreated roots, 92 proteins had different relative content. Bioinformatic tools such as STRING, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of network amongst the identified proteins. Results: From this analysis it was possible to evaluate that all of the identified proteins were functionally classified into several categories, mainly redox homeostasis, response to inorganic substances, energy metabolism, protein synthesis, cell trafficking, and division. Conclusion: In the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Activation of enzymes of the glycolytic pathway and up regulation of ribosomal protein indicated a stimulation in energy metabolism and protein synthesis. Regulation of the enzymes involved in redox homeostasis suggest a pivotal role of reactive oxygen species in the signaling and modulation of HS-induced responses

Comparative analysis of hyoscine in wild-type and in vitro grown Datura innoxia by high performance liquid chromatography

Purpose: To compare the hyoscine contents of Datura innoxia plant grown in the wild and that grown in vitro.Methods: For callus induction from leaf explants, 15 combinations of 2,4-dichlorophenoxy acetic acid, gibberellic acid, benzyl amino purine and kinetin were used. For regeneration of shoots, 8 combinations of BAP, kinetin and indole butyric acid (IBA) were tested. Different concentrations of IBA were used for rooting. The hyoscine contents of the wild and in vitro samples was initially identified by TLC and subsequently quantified by high performance liquid chromatography using hyoscine N-butyl bromide as standard.Results: For callus induction, 2,4-D at 2 mg/L produced maximum calli (96 %). Maximum shoots regeneration was obtained with 2 mg/L BAP and 1 mg/L kinetin. IBA at 0.5 mg/L induced good rooting. Substantial amount of hyoscine was detected in the extracts of in vitro grown plants through TLC and HPLC. Wild root, stem and leaves exhibited higher amounts (approx. 2 μg/mL) of hyoscine than the parts of in vitro grown plants. Green callus grown in vitro contained a maximum concentration of hyoscine (1.01 μg/mL) followed by leaf (0.82 μg/mL) and brown callus (0.432 μg/mL).Conclusion: The callus cultures of D. innoxia are capable of yielding good amounts of hyoscine, and therefore can be exploited to increase hyoscine production using chemical/hormone treatments on a large scale. Thus, this study provides a sustainable and efficient way for the sustainable production of a natural hyoscine product via in vitro-grown D. innoxia cultures.Keywords: Datura innoxia, Hyoscine, Callus, Hyoscine, Explant, Gibberellic acid, Benzyl amino purine, Kineti

Salt tolerance in indica rice cell cultures depends on a fine tuning of ROS signalling and homeostasis

<div><p>Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant’s response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant’s response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H₂O₂) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H₂O₂ burst along with controlled intracellular H₂O₂ and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO’s possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. <i>OsRbohA</i> and recently characterised OSCA family members i.e. <i>OsOSCA 1</i>.<i>2</i> and <i>OsOSCA 3</i>.<i>1</i> was done. Intracellular H₂O₂/NO levels displayed an interplay between Ca²⁺ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H₂O₂. Salt stress and ROS responsive TFs i.e. <i>OsSERF1</i> and <i>OsDREB2A</i> were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K⁺]/[Na⁺] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.</p></div

An Analysis 0f The Use 0f Speech Acts by English Language Teachers at College Level

The study aimed at analysing the use of speech acts by college level English language teachers in their classroom discussion. The objectives of the study were (i) to explore whether College level English language teachers in Pakistan employ speech acts in the class and (ii) to describe the kinds of speech acts employed by English language teachers at college level. The descriptive research design was used to conduct the study. Five male English language teachers with at least five years of experience in the field of English language teaching were selected as the participants of the study and an observation sheet was used to collect data in the form of utterances used by the teachers during their lectures. The research found that four types of speech acts were performed by English language teachers during their teaching practice and these included directives, representatives, commissives and expressives. The study concluded that the teachers’ awareness and effective use of speech acts may save the learners from misunderstanding during the interpretation of teachers’ intended messages and enhance the effectiveness of the teaching process

DISCURSIVE REPRESENTATION OF WOMEN IN CRIME NEWS STORIES OF PAKISTANI ENGLISH PRINT MEDIA

Media are a powerful source that reflects and mirrors societal attitude to culture. It also constructs and reconstructs our approaches and ideological inclinations. Pakistani media hold mirror up to the cultural influence on ideas and thoughts. The present study endeavors to explore the representation of women victims in crime news stories in Pakistani English newspapers. Data were collected from widely read main stream English newspapers and pragmatic approach was used to analyze linguistic choices which represent women. To analyze media discourse, Mill’s approach of Feminist Stylistics was used along with Corpus Linguistics. Data were quantified with the help of corpus software WMatrix (Rayson, 2008) to discover the frequency of lexical choices being used to represent women. The findings reveal that the female victims were mostly asymmetrically constructed and they were chiefly described in term of their age, social and marital status

DIRECTIVES IN L2: ANALYZING PRAGMATIC COMPETENCE OF PAKISTANI ENGLISH LEARNERS

Realization of directives namely ordering, commanding and asking effectively in English language is a problem for EFL speakers as they cannot recognize underlying pragmatic functions of speech acts (Beebe et al 1990 and Alam and Gill, 2016). L2 speakers usually look at the semantic meanings of the words which may cause communication breakdown as well as discourtesy in discourse. The research aims at finding similarities and differences in terms of using directives between Pakistani English learners (here on PELs) and native speakers (here on NSs). To achieve the objectives of the study quantitative method approach research design was utilized. The population for the study was native speakers from England and non-native speakers from Pakistan. The participants of the study were chosen through non-random purposive sampling technique and a total number of 80 samples (20 native British students studying in The Sheffield College, South Yorkshire England and 60 non-native Pakistani students of BS English studying in public-sector colleges of Lahore, Pakistan) were selected for collecting responses through DCTs. Data were analyzed per model of analysis of semantic formulae as proposed by Blum-Kalka and Olshtain (1984). The data were analyzed through SPSS. The results of the study were: (a) Pakistani EFL learners, with Punjabi as L1, were found deficient in terms of accuracy in uttering directives as compared to native speakers, (b) Pakistani EFL learners were found more direct as compared to native speakers, and (c) PELs were using less politeness strategies as compared to native speakers. The implications of the research are for the EFL learners, EFL teachers, curriculum designers, authors, and assessment purposes

Protein quantification and enzyme activity estimation of Pakistani wheat landraces.

Wheat is a major food grain in Pakistan having a prominent role in agriculture as well as the economic status of the country. In the current study, seeds of 99 wheat landraces were characterized for the quantification of seed storage proteins (Albumins, Globulin, Gliadins, and Glutenin), enzyme activities of antioxidant enzymes i.e. Ascorbate peroxidase (APX), Catalase (CAT), Superoxide dismutase (SOD), Peroxidase (POD), one hydrolytic enzyme Protease (PROT) and non-enzymatic antioxidant enzyme Ascorbic acid (AsA). The landraces were categorized into low, medium, and high based on protein concentration and enzymes activities/content. The majority of the landraces were placed in the medium category. However, for the AsA parameter majority of the landraces were placed in the low category. The highest concentration of total extracted protein (184.88±0.7 mg/g. wt.), globulins (21.35±0.43 mg/g. wt.) and glutenin (20±0.04 mg/g. wt.) as well as the high activity of SOD (303±16.80 Units/g. wt.), and Ascorbic acid (533±36.1 Units/g. wt.) was identified in the wheat landrace "11757" collected from district Panjgur (Balochistan). The wheat landrace "11760", collected from district Kech (Balochistan), contained the highest albumins concentration (65.42±0.02 mg/g. wt.) and highest activity for CAT (589.5±61.20 Units/g. wt.). The highest activity of POD (32341± 91.3) and PROT was observed in seeds of the wheat landrace "11618" collected from the Gilgit Baltistan region of Pakistan. The principal component analysis showed that the great variations existed for the tested parameters among the wheat landraces. The landraces with a high concentration of seed storage proteins and antioxidant enzyme activities can be used for breeding purposes to improve the nutrimental quality of wheat cultivars

Tissue microarray profiling and integrative proteomics indicate the modulatory potential of Maytenus royleanus in inhibition of overexpressed TPD52 in prostate cancers

Abstract Maytenus roylanus (MEM) is a plant with anti-proliferative effects against prostate cancer. We aimed to explore the mechanism of action of MEM in prostate cancer (PCa) by employing an in vitro global proteome approach to get useful information of various signaling pathways and effected genes to define the mechanism of MEM action in prostate cancer. We conducted a global proteome analysis of CWR22Rv1after treatment with methanolic extract of MEM. The result of the proteomic profiling of in vitro PCa cells demonstrated the reduction in tumor protein D52 (TPD52) expression after treatment with methanolic extract of MEM. Down-regulation of TPD52 expression at mRNA level was observed by MEM treatment in CWR22Rν1 and C4-2 cells in a dose-dependent fashion probably by cleavage of Caspase 3 and PARP, or by modulation of cyclin-dependent kinases in CWR22Rν1 and C4-2 cells. The progressive character of the TRAMP model demonstrates a chance to evaluate the potential of chemo-preventive agents for both initial and late stages of prostate cancer development, and induction in TPD52 protein expression with development as well as the progression of prostate cancer was observed in the TRAMP model. Analyses of the tissue microarray collection of 25 specimens confirmed the clinical significance of our findings identifying TPD52 as a potential marker for PCa progression. We determined that knockdown of TPD52 (CWR22Rν1 cells), a considerable downregulation was seen at the protein level. Downregulation of TPD52 inhibited the migration and invasive behavior of prostate cancer cells as observed. Moreover, we observed that the siRNA-TPD52 transfection of CWR22Rν1 cells resulted in tumor growth inhibition with a marked reduction in the secretion of prostate-specific antigen (PSA) in the serum. Intraperitoneal injection of MEM considerably slowed tumor growth in athymic mice, inhibited TPD52 expression, and caused a marked reduction in PSA levels of serum as demonstrated by immunoblot screening and immune-histochemical staining. This report illustrates a molecular overview of pathological processes in PCa, indicating possible new disease biomarkers and therapeutic targets