Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

Can Alternative Metabolic Pathways and Shunts Overcome Salinity Induced Inhibition of Central Carbon Metabolism in Crops?

The annual cost of lost crop production from exposure to salinity has major impacts on food security in all parts of the world. Salinity stress disturbs energy metabolism and knowledge of the impacts on critical processes controlling plant energy production is key to successfully breeding salt tolerant crops. To date, little progress has been achieved using classic breeding approaches to develop salt tolerance. The hope of some salinity researchers is that through a better understanding of the metabolic responses and adaptation to salinity exposure, new breeding targets can be suggested to help develop salt tolerant crops. Plants sense and react to salinity through a complex system of sensors, receptor systems, transporters, signal transducers, and gene expression regulators in order to control the uptake of salts and to induce tolerant metabolism that jointly leads to changes in growth rate and biomass production. During this response, there must be a balance between supply of energy from mitochondria and chloroplasts and energy demands for water and ion transport, growth, and osmotic adjustment. The photosynthetic response to salinity has been thoroughly researched and generally we see a sharp drop in photosynthesis after exposure to salinity. However, less attention has been given to the effect of salt stress on plant mitochondrial respiration and the metabolic processes that influence respiratory rate. A further complication is the wide range of respiratory responses that have been observed in different plant species, which have included major and minor increases, decreases, and no change in respiratory rate after salt exposure. In this review, we begin by considering physiological and biochemical impacts of salinity on major crop plants. We then summarize and consider recent advances that have characterized changes in abundance of metabolites that are involved in respiratory pathways and their alternative routes and shunts in terms of energy metabolism in crop plants. We will consider the diverse molecular responses of cellular plant metabolism during salinity exposure and suggest how these metabolic responses might aid in salinity tolerance. Finally, we will consider how this commonality and diversity should influence how future research of the salinity responses of crops plants should proceed.

Molecular cloning and in-depth bioinformatics analysis of type II ribosome-inactivating protein isolated from Sambucus ebulus.

Plant ribosome-inactivating proteins (RIPs) are N-glycosidases which inhibit protein synthesis through depurination of the ribosomal RNA sequence. Type II RIPs are heterodimer proteins which can bind to cell surfaces. The cytotoxicity of these RIPs is different. Sambucus spp. are a rich source of RIP proteins with different properties. In the present study, a type II RIP was isolated from S. ebulus plant that grows widely in the north of Iran, and different bioinformatics tools were used for the evaluation of physicochemical, functional and 3D protein characteristics. The results showed significant differences among isolated RIP and other Sambucus RIP proteins. The study of these differences can not only expand our insight into the functioning mechanisms of plant RIPs but also provide information about a novel RIP protein with potential biological applications.

Patient privacy: Awareness and attitudes of Iran University of Medical Sciences medical students.

Background: Respecting patients' privacy is an essential professional responsibility for physicians and other health team members. In this regard, this study investigates medical students' knowledge and attitude about confidentiality and disclosure of patients' information. Methods: In this cross-sectional study, 160 medical students of Iran University of Medical Sciences participated who were selected using stratified random sampling. Data were gathered using a valid and reliable self-report questionnaire. Student's knowledge and attitude toward medical confidentiality were assessed using self-administered and researcher-made questionnaires. Cronbach's alpha coefficients for knowledge and attitude levels were 79.7 and 82.2, respectively. Results: The average of medical students' responses to knowledge and attitude questions were 56.6% (9.6/ 17) and 55.3% (9.4 out of 17), respectively. On average, females had an acceptable attitude about 57.5% of the questions, whereas this was 50.9% for males. On average, females had an acceptable knowledge about 59.5% of the questions, whereas this was 50.6% for males. Therefore, female's attitudes and knowledge were more correct than their male counterparts (p<0.001). Conclusion: The low level of knowledge and attitude of medical students towards medical confidentiality indicates that revision of Iranian medical education curriculum to reinforce attention and knowledge of medical students on this issue to render appropriate care to patients is a necessity. Medical students' knowledge and attitude towards patient's confidentiality rights is not fulfilling.

Comparative proteomic analysis of salt-responsive proteins in canola roots by 2-DE and MALDI-TOF MS.

Salinity stress is a major abiotic stress that affects plant growth and limits crop production. Roots are the primary site of salinity perception, and salt sensitivity in roots limits the productivity of the entire plant. To better understand salt stress responses in canola, we performed a comparative proteomic analysis of roots from the salt-tolerant genotype Safi-7 and the salt-sensitive genotype Zafar. Plants were exposed to 0, 150, and 300 mM NaCl. Our physiological and morphological observations confirmed that Safi-7 was more salt-tolerant than Zafar. The root proteins were separated by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry was applied to identify proteins regulated in response to salt stress. We identified 36 and 25 protein spots whose abundance was significantly affected by salt stress in roots of plants from the tolerant and susceptible genotype, respectively. Functional classification analysis revealed that the differentially expressed proteins from the tolerant genotype could be assigned to 14 functional categories, while those from the susceptible genotype could be classified into 9 functional categories. The most significant differences concerned proteins involved in glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Fructose-bisphosphate aldolase, Phosphoglycerate kinase 3), stress (heat shock proteins), Redox regulation (Glutathione S-transferase DHAR1, L-ascorbate peroxidase), energy metabolism (ATP synthase subunit B), and transport (V-type proton ATPase subunit B1) which were increased only in the tolerant line under salt stress. Our results provide the basis for further elucidating the molecular mechanisms of salt-tolerance and will be helpful for breeding salt-tolerant canola cultivars.

Ion homeostasis, osmoregulation, and physiological changes in the roots and leaves of pistachio rootstocks in response to salinity.

Pistachio, one of the important tree nuts, is cultivated in arid and semi-arid regions where salinity is the most common abiotic stress encountered by this tree. However, the mechanisms underlying salinity tolerance in this plant are not well understood. In the present study, five 1-year-old pistachio rootstocks (namely Akbari, Badami, Ghazvini, Kale-Ghouchi, and UCB-1) were treated with four saline water regimes (control, 8, 12, and 16 dS m-1) for 100 days. At high salinity level, all rootstocks showed decreased relative water content (RWC), total chlorophyll content (TCHC), and carotenoids in the leaf, while ascorbic acid (AsA) and total soluble proteins (TSP) were reduced in both leaf and root organs. In addition, the total phenolic compounds (TPC), proline, glycine betaine, total soluble carbohydrate (TSC), and H2O2 content increased under salinity stress in all studied rootstocks. Three different ion exclusion strategies were observed in the studied rootstocks: (i) Na+ exclusion in UCB-1, because most of its Na+ is retained in the roots; (ii) Cl- exclusion in Badami, in which most of its Cl- remained in the roots; and (iii) similar concentrations of Na+ and Cl- were observed in the leaves and roots of Ghazvini, Akbari, and Kale-Ghouchi. Transport capacity (ST value) of K+ over Na+ from the roots to the leaves was more observable in UCB-1 and Ghazvini. Overall, the root system cooperated more effectively in UCB-1 and Badami for retaining and detoxifying an excessive amount of Na+ and Cl-. The results presented here provide important inputs to better understand the salt tolerance mechanism in a tree species for developing more salt-tolerant genotypes. Based on the results obtained here, the studied rootstocks from tolerant to susceptible are arranged as follows: UCB-1 > Badami > Ghazvini > Kale-Ghouchi > Akbari.

Proteomic analysis of soybean seedling leaf under waterlogging stress in a time-dependent manner.

Leaf is sensitive to environmental changes and exhibits specific responses to abiotic stress. To identify the response mechanism in soybean leaf under waterlogging stress, a gel-free/label-free proteomic technique combined with polyethylene glycol fractionation was used. Attenuated photosynthesis by waterlogging stress in the leaf of soybean seedlings was indicated from proteomic results. Defensive mechanisms such as reactive oxygen species (ROS) scavenging was also recognized. Cluster analysis revealed that proteins that exhibit characteristic dynamics in response to waterlogging were mainly related to photosynthesis. Among the identified photorespiration-related proteins, the protein abundance and enzyme activity of hydroxypyruvate reductase were transiently increased in control plants, but were clearly decreased in response to waterlogging stress. These results suggest that waterlogging directly impairs photosynthesis and photorespiration. Furthermore, hydroxypyruvate reductase may be a critical enzyme controlling the rate of photorespiration.

Comprehensive proteomic analysis of canola leaf inoculated with a plant growth-promoting bacterium, Pseudomonas fluorescens, under salt stress.

Plant growth-promoting bacteria can improve the tolerance of canola to salt stress. To better understand the effects of plant growth-promoting bacterium on the protein profiles of canola under salt stress condition, proteomics was performed. Salt-sensitive (Sarigol) and -tolerant (Hyola308) canola cultivars were inoculated with Pseudomonas fluorescens FY32, and the protein profiles of canola leaves were compared using a PEG-fractionation method. Cluster analysis of canola cultivars based on a stress tolerance index of several morphological parameters was used to confirm that Sarigol and Hyola308 were salt-sensitive and -tolerant cultivars, respectively. Using a gel-free proteomic technique, 154 and 94 proteins in Hyola308 and 100 and 144 proteins in Sarigol were uniquely identified in non-inoculated and bacterial-inoculated cultivars, respectively. By PEG fractionation, a total of 132 and 207 proteins were identified in non-inoculated and inoculated Hyola308, respectively. Notably, the abundance of copper/zinc superoxide dismutase 1 was significantly increased in inoculated Hyola308 under severe salt stress and decreased under moderate salt stress. In addition, the enzyme activity of delta-1-pyrroline-5-carboxylate synthase was significantly increased non-inoculated Hyola308 and the activity of succinate dehydrogenase was increased in inoculated Hyola308 leaves exposed to salt stress. Taken together, these results suggest that the bacterial inoculation of canola increases salt tolerance by inducing an increase in the abundance of proteins related to glycolysis, tricarboxylic acid cycle, and amino acid metabolism.

Proteomic analysis of canola root inoculated with bacteria under salt stress.

Plant-growth promoting bacteria can ameliorate the negative effects of salt stress on canola. To better understand the role of bacteria in canola under salt stress, salt-sensitive (Sarigol) and salt-tolerant (Hyola308) cultivars were inoculated with Pseudomonas fluorescens and protein profiles of roots were compared. Bacterial inoculation increased the dry weight and length of canola roots under salt stress. Using a gel-free proteomic technique, 55 commonly changed proteins were identified in Sarigol and Hyola308 roots inoculated with bacteria under salt stress. In both canola cultivars, proteins related to amino acid metabolism and tricarboxylic acid cycle were affected. Hierarchical cluster analysis divided the identified proteins into three clusters. Proteins related to Clusters II and III, which were secretion-associated RAS super family 1, dynamin-like protein, and histone, were increased in roots of both Sarigol and Hyola308 inoculated with bacteria under salt stress. Based on pathway mapping, proteins related to amino acid metabolism and the tricarboxylic acid cycle significantly changed in canola cultivars inoculated with or without bacteria under salt stress. These results suggest that bacterial inoculation of canola roots increases tolerance to salt stress by proteins related to energy metabolism and cell division. BIOLOGICAL SIGNIFICANCE: Plant-growth promoting bacteria as an emerging aid can ameliorate the negative effect of salt stress on canola. To understand the role of bacteria in canola under salt stress, salt sensitive Sarigol and tolerant Hyola308 cultivars were used. Dry weight and length of canola root were improved by inoculation of bacteria under salt stress. Using gel-free proteomic technique, 55 commonly changed proteins identified in Sarigol and Hyola308 inoculated with bacteria under salt stress. In both canola cultivars, the number of proteins related to amino acid metabolism and tricarboxylic acid cycle was more than other categories with higher change in protein abundance. Hierarchical cluster analysis divided into 3 clusters. Cluster II including secretion-associated RAS super family 1 and dynamin-like protein and Cluster III including histones H2A were increased by bacterial inoculation in both cultivars. Furthermore, pathway mapping highlighted the importance of S-denosylmethionine synthetase and malate dehydrogenase that decreased in canola inoculated with bacteria under salt stress. These results suggest that bacterial inoculation helps the canola to endure salt stress by modulating the proteins related to energy metabolism and cell division.

Inhibitory and cytotoxic activities of salvia officinalis L. Extract on human lymphoma and leukemia cells by induction of apoptosis.

PURPOSE: Salvia officinalis L., also known as Maryam Goli, is one of the native plants used to Persian medicinal herbs. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized crude methanol extracts prepared from Salvia officinalis L., on a non-Hodgkin's B-cell lymphoma (Raji) and human leukemic monocyte lymphoma (U937), Human acute myelocytic leukemia (KG-1A) and Human Umbilical Vein Endothelial (HUVEC) cell lines. METHODS: The effect of methanolic extract on the inhibition of cell proliferation and cytotoxic activity was evaluated by Dye exclusion and Micro culture tetrazolium test (MTT) cytotoxicity assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determined whether the mechanism involves induction of apoptosis or necrosis. RESULTS: The present results demonstrated that methanolic extract at 50 to 800 µg/ml dose and time-dependently suppressed the proliferation of KG-1A, U937 and Raji cells by more than 80% (p<0.01), with ascending order of IC50 values in 24: KG-1A (214.377 µg/ml), U937 (229.312 µg/ml) and Raji (239.692 µg/ml) when compared with a chemotherapeutic anticancer drug, paclitaxel (Toxol), confirming the tumour-selective cytotoxicity. The crude extract however did not exert any significant cytotoxic effect on normal cell line HUVEC (IC50>800 Ag/ml). Nucleosome productions in KG-1A, Raji and U937 cells were significantly increased respectively upon the treatment of Salvia officinalis L. extract. CONCLUSION: The Salvia officinalis L. extract was found dose and time-dependently inhibits the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous cell carcinoma.

Development of novel therapeutic modalities is crucial for the treatment of oral squamous cell carcinoma (OSCC). Recent scientific studies have been focused on herbal medicines as potent anti-cancer drug candidates. This study is the first to investigate the cytotoxic effects and the mechanism of cell death induced by grape seed extract (GSE) in oral squamous cell carcinoma (KB cells). MTT (3-(4,5-dimetylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) and trypan blue assays were performed in KB cells as well as human umbilical vein endothelial cells (HUVEC) were used to analyze the cytotoxic activity of GSE. Furthermore, the apoptosis-inducing action of the extract was determined by TUNEL, DNA fragmentation and cell death analysis. Statistical significance was determined by analysis of variance (ANOVA), followed by Duncan's test at a significance level of P≤0.05. The results showed apoptotic potential of GSE, confirmed by significant inhibition of cell growth and viability in a dose- and time- dependent manner without inducing damage to non-cancerous cell line HUVEC. The results of this study suggest that this plant contains potential bioactive compound(s) for the treatment of oral squamous cell carcinoma.

Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis.

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 µg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.

New insights into HLA class I association to Behçet's syndrome in Iranian Azari patients.

Behçet's syndrome (BS) is a chronic recurrent inflammatory disorder characterized by oral and genital ulcers and ocular inflammation. BS has a complex genetic etiology. To evaluate the influence of human leukocyte antigen (HLA) class I in BS susceptibility in Iranian Azari population, we studied 290 BS patients and 300 healthy controls. As expected, a high frequency of HLA-B5 was found. Remarkably, HLA-B35 frequency was higher in the patient than control group, and the frequency of HLA-B51, HLA-B52, and HLA-BW4 was significantly elevated. Thus, HLA-B5 and HLA-B35 seem to confer susceptibility to BD in Iranian Azari patients.

Behçet's syndrome in Iranian Azari people.

Behçet's Syndrome (BS) is a chronic recurrent multisystemic inflammatory disorder characterized by oral and genital ulcers, ocular inflammation. Behçet's syndrome has a complex genetic etiology. However, epidemiological studies recommend that genetic factors have a significant influence to its pathogenesis, alike to other autoinflammatory disorders. Epidemiological statistics, clinical records and HLA typing were studied in Iranian Azari patients with Behçet's syndrome. This investigation considered HLA associations with BS and HLA with certain clinical characteristics, age and sex in the (Tabriz) Iran which has an ethnically homogeneous population. HLA-A and HLA-B typing was performed in 290 BS patients, conforming to International Study Group criteria and in 300 blood donors, as controls. Patient records were retrospectively reviewed and patients reassessed clinically. HLA-B5, HLA-B35, HLA-51, HLA-B52 and HLA-CW4 presented significantly high frequencies in all patients. No other HLA type was associated. There was a significant HLA link with male sex in BS patients and Mean age (34 +/- 1.1) was determined. We present the frequency and correlation between Iranian Azari patients with Behçet's syndrome and particular HLA antigens. Ninety nine percent had mouth ulceration, 64% genital ulceration, 72% skin lesions and 52% ocular involvement. This study supports HLA-B5, HLA-B35, HLA-51, HLA-B52 and HLA-CW4 immunogenetic predisposition in an ethnically homogeneous (Iranian Azari) population.

Comparative proteomic analysis of canola leaves under salinity stress.

Although canola is a moderately salt-tolerant species, its growth, seed yield, and oil production are markedly reduced under salt stress, particularly during the early vegetative growth stage. To identify the mechanisms of salt responsiveness in canola, the proteins expressed in the second and third newly developed leaves of salt-tolerant, Hyola 308, and salt-sensitive, Sarigol, cultivars were analyzed. Plants were exposed to 0, 175, and 350 mM NaCl during the vegetative stage. An increase in the Na content and a reduction in growth were observed in the third leaves compared to the second leaves. The accumulation of Na was more pronounced in the salt-sensitive compared with the salt-tolerant genotype. Out of 900 protein spots detected on 2-DE gels, 44 and 31 proteins were differentially expressed in the tolerant and susceptible genotypes, respectively. Cluster analysis based on the expression level of total and responsive proteins indicated that the second leaves had a discriminator role between the two genotypes at both salinity levels. Using MS analysis, 46 proteins could be identified including proteins involved in responses to oxidative stress, energy production, electron transport, translation, and photosynthesis. Our results suggest that these proteins might play roles in canola adaptation to salt stress.