Plant Protein Improvement by Genetic Engineering: Use of Synthetic Genes.

The essential amino acid composition of plant foodstuffs is very important, especially in the developing countries where people very heavily depend on plant proteins from a single source. In these situations, it would be highly beneficial to \u27engineer\u27 the plant to produce proteins with a balanced essential amino acid content. Recombinant DNA technology and Agrobacterium-based vector systems offer a novel approach to modify plant proteins. Several attempts with some encouraging results have been made to express plant storage protein genes in transgenic plants. However, improvement of the quality of plant protein using these techniques is in an early stage. In this dissertation, the author tried to develop a system to improve the nutritional value of plant proteins using synthetic genes by utilizing the current techniques of recombinant DNA technology and Agrobacterium-based vector systems. Using a synthetic gene (HEAAE-DNA), which is 292 basepairs long and encodes a protein composed of about 80% essential amino acids, the author transformed potato plants using an A. rhizogenes vector system and tobacco plants using an A. tumefaciens vector system. Intact potato plants and tobacco plants were regenerated from hair roots infected with transformed A. rhizogenes and leaf-disks infected with transformed A. tumefaciens, respectively. Tubers from regenerated potato plants and leaves from regenerated tobacco plants were subjected to analysis for introduction and expression of this gene. Integration of the gene into the plant genome and its expression into mRNAs and HEAAE-proteins have been demonstrated in both cases using southern, northern and western analysis. Densitometric analysis of western blot data has shown that the HEAAE-proteins comprise up to 0.65% of the total plant proteins. The significant improvement of essential amino acid content of plant foodstuffs has not been achieved due to a low level of expression. However, a system has been developed to improve the nutritional value of plant foodstuffs using synthetic genes which, we hope, will eventually produce nutritionally complete plant proteins

Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

BACKGROUND: DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. RESULTS: We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. CONCLUSION: This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction

Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR

<p>Abstract</p> <p>Background</p> <p>The wild grass species <it>Brachypodium distachyon </it>(Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.</p> <p>Results</p> <p>A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST) databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR) using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (<it>UBC18</it>) was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (<it>Ubi4 </it>and <it>Ubi10</it>) was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (<it>SamDC</it>) ranked was most stable in plants grown under various environmental stresses.</p> <p>Conclusion</p> <p>This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.</p

Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells

RNA-dependent RNA polymerase 6 (RDR6) catalyses dsRNA synthesis for post-transcriptional gene silencing (PTGS)-associated amplification and the generation of endogeneous siRNAs involved in developmental determinations or stress responses. The functional importance of RDR6 in PTGS led us to examine its connection to the cellular regulatory network by analyzing the hormonal responses of RDR6 gene expression in a cultured cell system. Delivery of dsRNA, prepared in vitro, into cultured rice (Oryza sativa cv. Japonica Dongjin) cells successfully silenced the target isocitrate lyase (ICL) transcripts. Silencing was transient in the absence of abscisic acid (ABA), while it became persistent in the presence of ABA in growth medium. A transcription assay of the OsRDR6 promoter showed that it was positively regulated by ABA. OsRDR6-dependent siRNA(ICL) generation was also significantly up-regulated by ABA. The results showed that, among the five rice OsRDR isogenes, only OsRDR6 was responsible for the observed ABA-mediated amplification and silencing of ICL transcripts. We propose that ABA modulates PTGS through the transcriptional control of the OsRDR6 gene

EFFECT OF SPORTS TAPING APPLIED FUNCTIONAL CORRECTION GARMENT FOR ADOLESCENT IDIOPATHIC SCOLIOSIS IMPROVEMENT

The purpose of this research was to analyze the effect of wearing underwear type functional garment for the correction scoliosis by conducting a case study. Two patients wore the garment for 8–week. Cobb’s angle and EMG activities during a gait were measured before and after the treatment for the analysis. Both subjects showed that the activities of the right psoas, biceps femoris and gluteus medius were increased. The Cobb’s angle was decreased after 8-week period of wearing. These results matched with the purpose of developed functional correction wear that induce the right psoas muscle contraction and the left psoas muscle relaxation. It seems to be useful wear for adolescent scoliosis patients who avoid using orthosis, and to use special functional underwear to improve their posture

Impacts of Heavy Rain and Typhoon on Allergic Disease

AbstractObjectivesAllergic disease may be increased by climate change. Recent reports have shown that typhoon and heavy rain increase allergic disease locally by concentration of airborne allergens of pollen, ozone, and fungus, which are causes of allergic disease. The objective of this study was to determine whether typhoon and heavy rain increase allergic disease in Korea.MethodsThis study included allergic disease patients of the area declared as a special disaster zone due to storms and heavy rains from 2003 to 2009. The study used information from the Korea Meteorological Administration, and from the National Health Insurance Service for allergic diseases (asthma, allergic rhinitis, and atopic dermatitis).ResultsDuring a storm period, the numbers of allergy rhinitis and atopic dermatitis outpatients increased [rate ratio (RR) = 1.191; range, 1.150–1.232] on the sixth lag day. However, the number of asthma outpatients decreased (RR = 0.900; range, 0.862–0.937) on the sixth lag day after a disaster period. During a storm period, the numbers of allergic rhinitis outpatients (RR = 1.075; range, 1.018–1.132) and atopy outpatients increased (RR = 1.134; range, 1.113–1.155) on the seventh lag day. However, the number of asthma outpatients decreased to RR value of 0.968 (range, 0.902–1.035) on the fifth lag day.ConclusionThis study suggests that typhoon and heavy rain increase allergic disease apart from asthma. More study is needed to explain the decrease in asthma

COMPARISON OF GAIT ANALYSIS BETWEEN ADOLESCENT IDIOPATHIC SCOLIOSIS PATIENTS AND AGE MATCHED CONTROLS

The purpose of this study was to compare kinematic variables and surface EMG values between adolescent idiopathic scoliosis patients and age matched controls during a gait. Six male patients and five male healthy subjects were recruited for this study. Six cameras were used for 3D motion capture and selected joint angles were computed. Eight pairs of surface EMG electrodes were placed on latissimus dorsi, psoas, glutaeus medius, and biceps femoris. Results revealed that the scoliosis patients showed smaller hip joint angles compared to the values of the controls and vice versa for the trunk tilting angle. Small EMG activity of latissimus dorsi also found from the patient group. This meant that the scoliosis clearly influenced to the abnormal posture during a gait. Such results may be helpful to develop rehabilitation exercise or device

Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses

<p>Abstract</p> <p>Background</p> <p>Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.</p> <p>Results</p> <p>In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed <it>in vitro</it>. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (<it>P </it>< 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220⁺cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-A^d) were increased on CD11c⁺dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice.</p> <p>Conclusion</p> <p>These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.</p

Amifostine ameliorates recognition memory defect in acute radiation syndrome caused by relatively low-dose of gamma radiation

This study examined whether amifostine (WR-2721) could attenuate memory impairment and suppress hippocampal neurogenesis in adult mice with the relatively low-dose exposure of acute radiation syndrome (ARS). These were assessed using object recognition memory test, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, and immunohistochemical markers of neurogenesis [Ki-67 and doublecortin (DCX)]. Amifostine treatment (214 mg/kg, i.p.) prior to irradiation significantly attenuated the recognition memory defect in ARS, and markedly blocked the apoptotic death and decrease of Ki-67- and DCX-positive cells in ARS. Therefore, amifostine may attenuate recognition memory defect in a relatively low-dose exposure of ARS in adult mice, possibly by inhibiting a detrimental effect of irradiation on hippocampal neurogenesis