Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

BACKGROUND: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. METHODS: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. RESULTS: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. CONCLUSION: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.     

The vitamin B1, B2, B6, B12, and E contents of hard red spring wheat as influenced by fertilization and cultivar

To evaluate the effects of fertilization and cultivar on the content of vitamins B₁, B₂, B₆, B₁₂, and E in wheat, seven fertility treatments with three levels of N (0, 56, and 280 kg/ha), two levels of P (0 and 122 kg/ha), and two levels of K (0 and 465 kg/ha) were applied on split plots to two relatively unrelated hard red spring wheat (Triticum aestivum L.) cultivars. An analysis of grain vitamins revealed that fertilizer P and K applications increased grain B₁ and B₂ levels by 60 and 61%, respectively, in Waldron wheat. The same treatments decreased the contents of B₁ and B₂ in Era. Application of K and N and/or P to Era wheat resulted in a large decrease in vitamin B₁ and B₂ content. Vitamin B₆ was not influenced significantly by fertility treatments. Vitamin B₁₂ levels were not significantly different due to the fertilizer treatments. The two cultivars differed significantly only in vitamin B₁₂, Era being 52% higher than Waldron. Vitamins B₁ and B₂ varied together over all treatments and both cultivars (r=0.93).Contribution from the Department of Soils, North Dakota State University, Fargo, N.D. 58102. Journal Series No. 1196     

AT1 receptors activation enhances the expression of MMP-2, MMP-13 and VEGF but not MMP-9 in B16F10 melanoma cells

Melanoma is one of the most aggressive cancers of all solid tumors. The effect of angiotensin II on expression of three Matrix Metalloproteinases (MMPs) and Vascular Endothelial Growth Factor (VEGF) in B16F10 melanoma cells was evaluated. Also the blocking effect of losartan on angiotensin II induced effects was assessed. B16F10 murine melanoma cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 24 h prior to experiment the serum free medium was used. Angiotensin II (0 M, 10(-10) M, 10(-9) M or 10(-8) M) alone or in combination with Losartan (10(-6) M) in RPMI-1640 replaced the medium for experiments. After the incubation time (0, 1, 2, 6 and 12 h) cells were harvested using 0.05% (w/v) Trypsin and then recovered by centrifugation. The expression of MMP-2, MMP-13, MMP-9 and VEGF in B16F10 cell lysate was assessed by immunoblotting. Angiotensin II significantly enhanced the expression of MMP-2, MMP-13 and VEGF by concentrations as low as 0.1 nM. But angiotensin II could not stimulate any significant increase in MMP-9 expression by angiotensin II in B16F10 cells. Losartan abolished the enhancing effect of every concentration of angiotensin II on MMP-2, MMP-13 and VEGF expression completely and in all incubation times. As a result, angiotensin II through activation of AT1 receptors can stimulate the expression of MMP-2, MMP-13 and VEGF in B16F10 melanoma cells. This is an important conclusion because of the importance of these factors in melanoma invasiveness and the possible important role that angiotensin receptor blockers may play as cancer medications.     

大豆品种安豆1498对大豆疫霉菌株PsJS2的抗性遗传分析及基因定位

为发掘新的抗病基因以增强大豆对疫霉根腐病的抗性,本研究以大豆品种安豆1498和Williams为材料,采用下胚轴创伤接种法鉴定二者对Race1、Race3、Race4、NKI、USAR2、Ps41-1、PsMC1和PsJS2等8个大豆疫霉菌株的抗性;对安豆1498与Williams杂交组合获得167个F2:3代家系群体...     

抗虫抗除草剂转基因水稻B1C893的获得与鉴定

利用农杆菌介导法将Cry1Ca#和Bar基因转化水稻恢复系R893,获得6株再生植株。经过分子鉴定,发现Bar基因和Cry1Ca#基因成功整合到水稻基因组并稳定表达。测定其中单拷贝且可育的转基因株系苗期不同器官中Cry1Ca蛋白的表达量,发现根系中最低而叶片中最高,其中最高的第6号株系叶片中含量达到35.07μg/g。获得的转基因株系对除草剂草铵膦和稻纵卷叶螟均具有良好的抗性。     

施用PL-2对油菜产量及主要性状的影响

在油菜抽薹初期叶面喷施生长调节剂PL-2 1 500mL/hm2,可使植株矮化25.2cm,各叶节间长度趋于均衡,防倒效果明显.薹茎下部表皮开裂减少,有利于预防菌丝侵染,增强抗病能力,菌核病发病率比对照减少30.3个百分点.PL-2有利于改善油菜植株冠层结构,提高光合效率,菜籽产量比未喷PL-2对照增产9.23%～13.28%,达显著水平.     

Human leukocyte antigen-G expression on dendritic cells induced by transforming growth factor-beta1 and CD4+ T cells proliferation

Background: During antigen capture and processing, mature dendritic cells (DC) express large amounts of peptide-MHC complexes and accessory molecules on their surface. DC are antigen-presenting cells that have an important role in tolerance and autoimmunity. The transforming growth factor-beta1 (TGF-Beta1) cytokine has a regulatory role on the immune and non-immune cells. The aim of this study is to evaluate the effect of TGF-Beta1 on the induction of human leukocyte antigen-G (HLA-G) expression on the DC which is derived from monocyte. Methods: In this study, we evaluated the effect of TGF-Beta1 in induction HLA-G expression on the monocyte-derived DC by flowcytometry and then CD4+ T cell proliferative responses in the presence of DC-treated TGF-Beta1 was studied. Results: The results of this study showed that DC bearing HLA-G down-regulated activation of CD4+ T cells and production of IL-6 and IL-17 in comparison with control (P<0.05). Conclusion: It is concluded that TGF-Beta1 has an important regulatory role in CD4+ T cell proliferation by increasing HLA-G on DC and these cells can probably prevent unexpected immune responses in vivo.     

Influence of Iron Deficiency Anemia on Hemoglobin A1C Levels in Diabetic Individuals with Controlled Plasma Glucose Levels

Introduction: Hemoglobin A1C (HbA1c) reflects patient’s glycemic status over the previous 3 months. Previous studies have reported that iron deficiency may elevate A1C concentrations, independent of glycemia. This study is aimed to analyze the effect of iron deficiency anemia on HbA1c levels in diabetic population having plasma glucose levels in control. Methods: Totally, 120 diabetic, iron-deficient anemic individuals (70 females and 50 males) having controlled plasma glucose levels with same number of iron-sufficient non-anemic individuals were streamlined for the study. Their data of HbA1c (Bio-Rad D-10 HPLC analyzer), ferritin (cobas e411 ECLIA hormone analyzer), fasting plasma glucose (FPG, Roche Hitachi P800/917 chemistry analyzer), hemoglobin (Beckman Coulter LH780), peripheral smear examination, red cell indices, and medical history were recorded. Statistical analysis was carried out by student’s t-test, Chi-square test, and Pearson’s coefficient of regression. Results: We found elevated HbA1c (6.8 ± 1.4%) in iron-deficient individuals as compared to controls, and elevation was more in women (7.02 ± 1.58%). On further classification on the basis of FPG levels, A1C was elevated more in group having fasting glucose levels between 100-126 mg/dl (7.33 ± 1.55%) compared to the those with normal plasma glucose levels (<100 mg/dl). No significant correlation was found between HbA1c and ferritin and hemoglobin. Conclusion: This study found a positive correlation between iron deficiency anemia and increased A1C levels, especially in the controlled diabetic women and individuals having FPG between 100-126 mg/dl. Hence, before altering the treatment regimen for diabetic patient, presence of iron deficiency anemia should be considered. Key Words: Iron deficiency anemia, Hemoglobin A1C (HbA1c), Diabetes     

Effects of melophlins on colony formation of Chinese hamster V79 cells and IL-8 production in PMA-stimulated HL-60 cells

We have recently isolated four new melophlins P (1), Q (2), R (3), and S (4) together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11) from two marine sponges of the genus Melophlus collected in Palau. In this study, the influence of these compounds on the colony formation of Chinese hamster V79 cells and the production of IL-8 in PMA-stimulated HL-60 cells were examined. These 11 compounds did not show any effect on IL-8 production. The EC₅₀ values of compounds 2, 3, 4, 5, 7, 9, 10, and 11 against V79 cells were 44.0, 13.3, 16.7, 27.2, 19.8, 8.5, 23.1, and 9.6 μM, respectively. The linear-chain-type compounds (1, 6, and 8) were not active against V79 cells at 50 μM. Although the growth inhibitory activity of these melophlins was not remarkable, some structure-activity relationships of these compounds against V79 and murine leukemia L1210 cells were observed.     

Verification of Glyphosate Resistance,Lepidopteran Resistance and Wide Compatibility of Male Sterile Line E1C4008S in Rice

Weeds and insect pests are two important biotic stresses resulting in yield loss in rice, and wide compatibility is the essential characteristic of breeding inter-subspecific hybrid rice. This study focused on glyphosate resistance, lepidopteran resistance and wide compatibility as well as identification of molecular and some agronomic characteristics of transgenic male sterile line E1 C4008 S. The results indicated that glyphosate resistance gene Epsps# and lepidopteran resistance gene Cry1 ca~# were transferred into japonica wide compatibility male sterile line 4008 S by Agrobacterium-mediated method, and four independent transformation events named E1 C4008 S-1, E1 C4008 S-2, E1 C4008 S-3 and E1 C4008 S-4 were obtained, in which E1 C4008 S-3 and E1 C4008 S-4 were of single copy insertion. The EPSPS protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf stem root, and ranged from 300.58 to 1410.69 μg/g at the tillering stage. The glyphosate tolerable concentration(dosage) of E1 C4008 S-3 and E1 C4008 S-4 reached at least 16 g/L(54.42 kg/hm2), and the seeds of E1 C4008 S-4 can germinate normally on the medium containing 1 g/L glyphosate. The CRY1 C protein contents of E1 C4008 S-3 and E1 C4008 S-4 in different organs were significantly different both in descending order of leaf stem root, and ranged from 0.62 to 2.43 μg/g at the tillering stage. The larvae mortalities of rice leaf rollers fed on leaves of E1 C4008 S-3 and E1 C4008 S-4 for 5 d were 95.35% and 97.77%, respectively, while the average mortalities of silkworms fed with protein extracts from leaves of E1 C4008 S-3 and E1 C4008 S-4 reached 94.55% and 83.64%, respectively. The results suggested that wide compatibility and evaluated agronomic traits of E1 C4008 S-4 were not significantly changed by insertion of the exogenous genes. Overall, a novel male sterile germplasm with glyphosate resistance, lepidopteran resistance and wide compatibility was verified to be developed in rice.     

Unusual Structures and Cytotoxicities of Chitonoidosides A,A1, B,C, D,and E,Six Triterpene Glycosides from the Far Eastern Sea Cucumber Psolus chitonoides

Six new triterpene tetra-, penta- and hexaosides, chitonoidosides A (1), A₁ (2), B (3), C (4), D (5), and E (6), containing one or two sulfate groups, have been isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from the depth of 100–150 m. Three of the isolated compounds (1, 3 and 6) are characterized by the unusual aglycone of new type having 18(20)-ether bond and lacking a lactone in contrast with wide spread holostane derivatives. Another unexpected finding is 3-O-methylxylose residue as a terminal unit in the carbohydrate chains of chitonoidosides B (3), C (4), and E (6), which has never been found before in the glycosides from holothurians belonging to the Psolidae family. Moreover, this monosaccharide is sulfated in the compound 4 into unprecedented 3-O-methylxylose 4-O-sulfate residue. Chitonoidoside C (4) is characterized by tetrasaccharide moiety lacking a part of the bottom semi-chain, but having disaccharide fragment attached to C-4 of Xyl1. Such architecture is not common in sea cucumber glycosides. Cytotoxic activities of the compounds 1–5 against mouse and human erythrocytes and human cancer cell lines: adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and leukemia promyeloblast HL-60 cells were studied. The cytotoxic effect of chitonoidoside d (5) was the most significant in this series due to the presence of pentasaccharide disulfated sugar chain in combination with holostane aglycone. Surprisingly, the glycosides 1 and 3, comprising the new aglycone without γ-lactone, demonstrated similar activity to the known compounds with holostane aglycones. Chitonoidoside C (4) was less cytotoxic due to the different architecture of the carbohydrate chain compared to the other glycosides and probably due to the presence of a sulfate group at C-4 in 3-O-MeXyl4.     

Calyculins and Related Marine Natural Products as Serine-Threonine Protein Phosphatase PP1 and PP2A Inhibitors and Total Syntheses of Calyculin A,B, and C

Calyculins, highly cytotoxic polyketides, originally isolated from the marine sponge Discodermia calyx by Fusetani and co-workers, belong to the lithistid sponges group. These molecules have become interesting targets for cell biologists and synthetic organic chemists. The serine/threonine protein phosphatases play an essential role in the cellular signalling, metabolism, and cell cycle control. Calyculins express potent protein phosphatase 1 and 2A inhibitory activity, and have therefore become valuable tools for cellular biologists studying intracellular processes and their control by reversible phosphorylation. Calyculins might also play an important role in the development of several diseases such as cancer, neurodegenerative diseases, and type 2-diabetes mellitus. The fascinating structures of calyculins have inspired various groups of synthetic organic chemists to develop total syntheses of the most abundant calyculins A and C. However, with fifteen chiral centres, a cyano-capped tetraene unit, a phosphate-bearing spiroketal, an anti, anti, anti dipropionate segment, an α-chiral oxazole, and a trihydroxylated γ-amino acid, calyculins reach versatility that only few natural products can surpass, and truly challenge modern chemists’ asymmetric synthesis skills.     

Influence of the growth retardant B 995 on growth and yield of potatoes

Summary Stem elongation in a potato crop can be reduced by spraying the growth retardant B 995 on the foliage. This treatment generally did not change the total dry matter production. Only in the last period of growth did B 995-treated plots show a higher total dry weight than the untreated ones. This was caused by a slightly later senescence of the treated plots. The B 995 treatment caused a marked change in the dry matter distribution: stem weight was decreased and tuber weight increased, but leaf weight was generally not influenced. With plants showing considerable vegetative growth, these effects resulted in an increased tuber yield. High nitrogen plots treated with B 995 combined a long productive periode with a favourable dry matter distribution, producing a very high tuber yield at the end of the growing period.     

新型植物生长调节剂HHPS-1、HHPS-2对马铃薯产量及品质影响的研究

为了将从俄罗斯引进的HHPS-1、HHPS-2两种植物生长调节剂应用到马铃薯生产中,本人对供试材料马铃薯品种克新16号进行了药剂的5种处理,在2004,2005年进行了田间比较试验,并对试验结果进行了统计分析。结果表明:两种试剂对其产量和品质均产生影响。HHPS-2对晚疫病有预防和控制作用,相反HHPS-1则不明显。两种植物生长调节剂均可使供试品种大、中薯率、淀粉含量提高,单位面积产量增加。     

Season-long expression of Cry1Ac and Cry2Ab proteins in Bollgard II cotton in Australia

Bollgard II cotton has been grown commercially in Australia since 2003 for control of the primary target species Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) Larvae of both species have been reported to survive at low frequencies on Bollgard II with larvae >8 mm recorded in between 7 and 18% of the area planted to Bollgard II cotton between 2005/06 and 2007/08. F₁ and F₂ tests have shown that this is not due to the presence of resistance genes in the surviving larvae. To understand if fluctuations in the expression of the Cry proteins in Bollgard II allow some larvae to survive, plant tissue samples were taken from five Bollgard II cultivars throughout the growing season within fields and from different farms within a production region between 2007 and 2010. The data indicate that the expression of both Cry proteins is similar to the known resistance-monitoring diagnostic concentrations and relatively uniform between fields within a farm and between farms within a region, with less than one-third of the tests at this level of variation being significant. However, there were intra-seasonal changes in expression of both Cry proteins and differences in expression between plant structures and between cultivars for both Cry proteins. Further work is needed to establish if this variation in Cry protein content in Bollgard II cotton affects the control of Helicoverpa spp. in the field or whether plant-physiological and pest-behavioural factors underlie the occasional occurrence of Helicoverpa larval survival on Bollgard II cotton.     

OsSCE1 Encoding SUMO E2-Conjugating Enzyme Involves in Drought Stress Response of Oryza sativa

Small ubiquitin-like modifier (SUMO)-conjugating enzymes are involved in post-translational regulatory processes in eukaryotes, including the conjugation of SUMO peptides to protein substrate (SUMOylation). SUMOylation plays an important role in improving plant tolerance to abiotic stress such as salt, drought, heat and cold. Herein, we reported the isolation of OsSCE1 (LOC_Os10g39120) gene encoding a SUMO-conjugating enzyme from rice (Oryza sativa cv. Nipponbare) and its functional validation in response to drought stress. The E2 enzyme, OsSCE1, is one of three key enzymes involved in the conjugation of SUMO to its target proteins. Activated SUMO is transferred to the cysteine of an E2 enzyme and then to the target lysine residue of the substrate, with or without the help of an E3 SUMO ligase. Expression of OsSCE1 was strongly induced by polyethylene glycol 6000 (PEG6000) treatment, which suggested OsSCE1 may be involved in the drought stress response. Overexpression of OsSCE1 (OsSCE1-OX) in Nipponbare reduced the tolerance to drought stress. Conversely, the drought tolerance was slightly improved by the knockdown of OsSCE1 (OsSCE1-KD). These results were further supported by measurement of proline content in OsSCE1-OX and OsSCE1-KD transgenic lines under induced drought stress, which showed OsSCE1-KD transgenic lines accumulated higher proline content than the wild type, whereas OsSCE1-OX line had lower proline content than the wild type. These findings suggested OsSCE1 may play a role as a negative regulator in response to drought stress in rice.     

非1B/1R和1B/1R类型K型小麦雄性不育系幼穗部分生理指标的变化

为了揭示非1B/1R及1B/1R类型K型小麦雄性不育系雄性败育的生化机制,在不同发育时期对K型非1B/1R小麦雄性不育系KTSP3314A及同型保持系TPS3314B与K型1B/1R小麦雄性不育系K3314A及同型保持系3314B幼穗中游离氨基酸总量、可溶性蛋白含量、可溶性糖和淀粉含量进行了测定.结果表明,非1B/1R类型的K型不育系幼穗的这些生理指标与1B/1R类型K型小麦雄性不育系的变化不同.1B/1R类型不育系除可溶性糖含量在整个发育时期与其同型保持系差别不明显外,其它物质的含量在单核小孢子时期均差异明显;非1B/1R类型不育系这四种物质含量在二核小孢子时期均差异较大.由此说明,这两类K型不育系的雄性败育进程有所不同,非1B/1R类型K型不育系的雄性败育时间晚于1B/1R类型K型不育系,并在雄性败育的过程中伴随着部分生理指标的变化.     

非1B/1R和1B/1R 类型小麦K型雄性不育系穗长和小穗数性状的遗传分析

为明确非1B/1R和1B/1R 类型小麦K 型雄性不育系穗长和小穗数的遗传规律,利用非1B/1R类型KTSP732A和1B/1R类型K3314A 两种K型不育系材料,采用主基因+多基因混合遗传模型分析法,对穗长和小穗数两类性状进行单世代和多世代混合分离分析.结果显示,非1B/1R类型KTSP732A和1B/1R类型K3314A穗长性状均符合加性-显性-上位性多基因模型(C-0),无主基因存在;非1B/1R类型KTSP732A的小穗数性状符合两对等显性主基因+加性-显性多基因混合模型(E-6),主基因遗传率为40.7%;而1B/1R类型K3314A的控制小穗数性状基因类型与非1B/1R不同,该类型无主基因存在,也符合加性-显性-上位性多基因模型(C-0).     

红光和蓝光对甘蓝型油菜黄籽和黑籽粒色的影响

在甘蓝型黄籽和黑籽油菜种子发育时期用红光和蓝光处理植株，待种子成熟后，分别测定其种皮中的PAL活性及蛋白质，多酚，花色素和黑色素的含量，结果表明，蓝光可降低PAL活性，增加蛋白质含量、减少多酚，花色素和黑色素的合成，PAL活性对甘蓝型油菜种皮色泽有影响，且对黄籽的影响更大。