Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

The green fluorescent protein (GFP) of jellyfish (_Aequorea victoria_) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (_hpt_) as a selective marker and a modified glycinin (11S globulin) gene (_V3-1_) as a target. However, sGFP(_S65T_) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(_S65T_) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(_S65T_) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(_S65T_) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system

Effect of Treadmill Training With Eyes Open and Closed on Knee Proprioception, Functional Balance and Mobility in Children With Spastic Diplegia

Objective To investigate the effect of treadmill training with eyes open (TEO) and closed (TEC) on the knee joint position sense (JPS), functional balance and mobility in children with spastic diplegia. Methods Forty-five children with spastic diplegia aged 11–13 years participated in this study. They were randomly assigned to three groups of equal number. The control group (CON) underwent designed physical therapy program whereas, the study groups (TEO and TEC) underwent the same program, in addition to treadmill gait training with eyes open and closed, respectively. Outcome measures were the degree of knee joint position error, functional balance and mobility. Measurements were taken before and after 12 weeks of intervention. Results After training, the three groups showed statistically significant improvement in all measured outcomes, compared to the baseline with non-significant change in the knee JPS in the CON group. When comparing posttreatment results, the TEC group showed greater significant improvement in all measured outcomes, than the TEO and CON groups. Conclusion Treadmill training with eyes open and closed is effective in rehabilitation of children with diplegia, but blocked vision treadmill training has more beneficial effect

Establishment of in vitro fast-growing normal root culture of Vernonia amygdalina - a potent African medicinal plant

Fast-growing normal root culture of Vernonia amygdalina, a potent African medicinal plant was established from leaf explants of in vitro raised shoot induced from the stem nodal segments on murashige and skoog (MS) medium containing 0.5 mg l-1 6-benzylaminopurine (BA) in combination with 0.5 mg l-1 naphthalene acetic acid (NAA). In vitro raised plantlets were maintained on MS agar medium and sub cultured at 4 weeks interval and used as leaf explant source. Explants were cultured on halfstrengthMS medium supplemented with different concentrations of Indole-3-acetic acid (IAA), indole-3- butyric acid (IBA) and NAA. Basal medium supplemented with IBA at 0.25 and 2.0 mg l-1 and under 16photoperiod condition favoured induction of the longest root (2.7 ± 1.1 cm) and highest number of roots/explant (38.3 ± 1.1) respectively. After 6 weeks well established roots were separated. Fresh root tissue, in amount of a 100 mg were cultured in 50 ml full-strength MS liquid medium supplemented with 2.0 mg l-1 IBA and under continuous agitation (80 rpm). The biomass of root culture was increased to 2.1949 g after 5 weeks of culture. The root culture was maintained up to 6 weeks. The protocoldeveloped in this study provides a basis for adventitious root induction and for further investigation of medicinally active constituents of this elite medicinal plant

Establishment of the Regeneration System for Vicia faba L.

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba . Two Egyptian faba bean cultivars \u27Giza 2\u27 and \u2724 Hyto\u27 were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean

The Interactions of the Largest Subunit of RNA Polymerase II with Other Cellular Proteins: a Bioinformatic Approach

The function of a protein is governed by its interaction with other proteins inside a cell. Therefore, it is important to identify the interacting partners of a particular protein to decipher its function. The protein interaction networks are generally determined by bioinformatic as well as experimental methodologies such as yeast two hybrid, mass spectrometry, immunoprecipitation, and fluorescence resonance energy transfer assays. Here, we have analyzed bioinformatically the interactions of Rpb1p (the largest subunit of RNA Polymerase II) with other proteins in yeast, using Cytoscape software and Biogrid/Biomart database. We find that Rpb1p interacts with a large number of proteins involved in mRNA synthesis, processing, export, and other cellular processes. These results validate the application of such bioinformatic approach to determine the interactome for other cellular proteins

A Protein Allergen Microarray Detects Specific IgE to Pollen Surface, Cytoplasmic, and Commercial Allergen Extracts

Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy

PROTECTIVE EFFECT OF MORINGA OLEIFERA SEED EXTRACT ON CISPLATIN INDUCED NEPHROTOXICITY IN RATS

Objective: The main objective of this study is to investigate the antioxidant and nephroprotective efficacy of moringa oleifera seed extract (MOSE) against cisplatin which induced acute renal injury. Methods: Forty male Wister rats were equally segregated into 4 groups (10 rats per group): group I (0.5 ml of sterile saline orally), group II (200 mg MOSE/kg b. wt orally for 10 consecutive days), group III (7.5 mg cisplatin/kg b. wt/intraperitonially as a single dose on the 5th day of the experiment) and group IV (200 mg moringa oleifera seed extract (MOSE)/kg orally for 10 d followed by 7.5 mg cisplatin/kg body weight/intraperitonially once as a single dose on the 5th day of the experiment. Serum biochemical analysis of renal biomarkers (urea, uric acid, and creatinine), oxidative stress markers (malondialdehyde [MDA]), a crucial antioxidant enzyme (catalase) and the expression of renal activity interleukin (IL)-6, (IL)-10 and Tumer necrotic factor (TNF-α) mRNA were determined. Histopathological examination of renal tissue was done. Results: Cisplatin induced renal damage, increased renal biomarkers (urea, creatinine and uric acid)(375.87±1.65, 5.238±0.25, 4.47±0.25). Tissue concentrations of malondialdehyde, IL-6 and TNF-α.(387.56±0.97, 2.188±0.20, 3.06±0.27)compared to control group(140.58±1.25,0.938±0.017, 1.24±0.17), (163.99±1.34, 1.008±0.05, 0.982±0.026) Moreover, cisplatin induced significantly down-regulation of anti-inflammatory (IL-10) and catalase (0.780±0.47, 1.62±0.06) compared to control one (1.010±0.02, 3.12±0.11),. The histopathological examination showed renal tissue damage and degeneration of tubules in the cortical portion in cisplatin group. However, interestingly concurrent adminsteration of the MOSE with cisplatin can alleviated the renal damage, oxidative stress and renal toxicity caused by cisplatin. Conclusion: These results suggest that the antioxidant and the anti-inflammatory effects of MOSE alleviate the cisplatin-induced nephrotoxicity

Prevalence of Coronary Artery Lesion(S) in Pati ents Aged 40-50 Years Undergoing Rheumatic Valvular Surgery

Objective: To determine the prevalence of coronary artery disease in patients undergoing valve surgery for rheumatic heart disease between age 40-50 years, usefulness, and indication of pre-operative coronary angiography. Methods: This is an observational prospective study that took place in 2 hospitals (National Heart Institute and Nasser Institute) within the period starting from January 2013 to January 2015. We included 454 rheumatic patients that were admitted for elective primary mitral, aortic or double valve surgery, and that had a coronary angiogram in their regular preoperative workup. All coronary angiographies were performed by injecting right and left coronaries by using 80-100 ml of iodinated contrast to obtain the standard views of both right and left coronaries using Philips or Siemens machines in both hospitals. Coronary artery disease (CAD) of 50% is considered to be a positive finding. Results: There was no correlation between rheumatic heart disease in this age group and CAD as only 1.76% had the significant stenosis. Male gender, family history of CAD, age above 45yrs, hypertension, and smoking showed significant correlation with the CAD in this study. Conclusion: Our results suggest that the overall prevalence of coronary artery disease in patients undergoing rheumatic valve surgery in our population is not comparable with the prevalence reported in international data. So, multicenter studies are needed in developing countries to set their own guidelines. Therefore, our study can be the nucleus for these guidelines in our country

Recovery of herbicide-resistant Azuki bean [Vigna angularis (Wild.), Ohwi & Ohashi] plants via Agrobacterium-mediated transformation

Transgenic azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] plants expressing the hygromycin phosphotransferase (hpt), green fluorescent protein (sgfp) and phosphinothricin acetyltransferase (bar) genes were obtained by Agrobacterium- tumefacients - mediated transformation. A total of 210 epicotyl explants were inoculated with A. tumefaciens strain EHA105, harboring the binary plasmid pZHBG on MS co-cultivation medium supplemented with 100 mM acetosyringone and 10 mg/l of BA. Following selection on MS medium with 15 mg/l of hygromycin, the regenerated adventitious shoots that formed on the induced calli were further screened for sgfp expression before transferred to rooting medium. 31 transgenic plants were obtained with transformation frequency of 14%. The presence of transgenes in transformed azuki bean plants was confirmed by polymerase chain reaction (PCR) and southern blot analysis. Transcription of the bar and hpt genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis. sgfp- positive transgenic plants exhibited functional expression of the bar gene as determined by assaying for resistance to bialaphos applied directly to leaves. This result demonstrates the feasibility of introducing potentially useful agronomic traits into azuki bean through genetic engineering. Key Words: Agrobacterium tumefaciens, bar gene, bialaphos, transgenic, Vigna angulazris. African Journal of Biotechnology Vol.4(1) 2005: 61-6

Efficient production of transgenic soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic (WSS) method

The present study was designed to evaluate the transformation efficiency and proof the capability of whisker supersonic (WSS) method as an alternative option for soybean (Glycine max [L] Merrill)transformation. We compared soybean transformation efficiency obtained by WSS-mediated with that of particle bombardment transformation by carrying out molecular analysis of the T0 plants in two independent experiments. For this, we used for both transformation techniques the same genotype, the same plasmid and the same selection method. To assess the efficiency of soybean genetictransformation, we evaluated the efficiency of multi gene transformation by the selection with hygromycin and the expression of green fluorescent protein [sGFP (S65T)] resulted from both techniques. Regenerable embryogenic cells were induced from immature cotyledons of soybean c.v Jack on MSD40 media within 3 weeks then proliferated on FN lite liquid media and engineered with pUHG gene construct through both WSS and particle bombardment-mediated transformation. The pUHG was constructed with pUC 19 and contain the hpt gene conferring resistance to hygromycin as a selective marker and sGFP(S65T) as a reporter gene. Fluorescence microscopy screening after the selection of hygromycin, identified the clearly expression of sGFP(S65T) in the transformed soybean embryos. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) andSouthern blot analysis. The average transformation efficiency achieved with WSS was higher than that obtained by particle bombardment and hence it may represent an alternative method for soybeantransformation