Vibration parameters affecting vibration-induced reflex muscle activity

Purpose: To determine vibration parameters affecting the amplitude of the reflex activity of soleus muscle during low-amplitude whole-body vibration (WBV).

Materials and methods: This study was conducted on 19 participants. Vibration frequencies of 25, 30, 35, 40, 45, and 50?Hz were used. Surface electromyography, collision force between vibration platform and participant’s heel measured using a force sensor, and acceleration measured using an accelerometer fixed to the vibration platform were simultaneously recorded.

Results: The collision force was the main independent predictor of electromyographic amplitude.

Conclusion: The essential parameter of vibration affecting the amplitude of the reflex muscle activity is the collision force.     

海洋来源节菱孢菌ZSDS1-F3中PKS-NRPS类天然产物挖掘

【目的】以基因组信息为指导,定向激活海洋来源真菌Arthrinium arundinisZSDS1-F3中沉默的聚酮合成酶-非核糖体肽合成酶(PKS-NRPS)类生物合成基因簇,鉴定次级代谢产物结构。【方法】通过启动子工程和异源表达的策略激活实验室培养条件下沉默或低表达的生物合成基因簇,实现目标化合物的分离,通过HR-ESI-MS和NMR数据分析鉴定产物结构,结合基因重组和生物信息学分析结果推导化合物的生物合成途径。【结果】依据基因组生物信息学分析,从海洋来源真菌A. arundinis ZSDS1-F3中选取一个编码PKS-NRPS类次级代谢产物的生物合成基因簇开展研究,在宿主Aspergillus nidulansA1145中实现了基因簇的异源表达,从中分离到2个新化合物,并推导了其生物合成途径。【结论】基因组信息指导下的天然产物挖掘,可以目标明确地分离产物,加快真菌中新颖天然产物的发现步伐。     

Computational modeling of human coreceptor CCR5 antagonist as a HIV-1 entry inhibitor: using an integrated homology modeling,docking, and membrane molecular dynamics simulation analysis approach

Chemokine receptor 5 (CCR5) is an integral membrane protein that is utilized during human immunodeficiency virus type-1 entry into host cells. CCR5 is a G-protein coupled receptor that contains seven transmembrane (TM) helices. However, the crystal structure of CCR5 has not been reported. A homology model of CCR5 was developed based on the recently reported CXCR4 structure as template. Automated docking of the most potent (14), medium potent (37), and least potent (25) CCR5 antagonists was performed using the CCR5 model. To characterize the mechanism responsible for the interactions between ligands (14, 25, and 37) and CCR5, membrane molecular dynamic (MD) simulations were performed. The position and orientation of ligands (14, 25, and 37) were found to be changed after MD simulations, which demonstrated the ability of this technique to identify binding modes. Furthermore, at the end of simulation, it was found that residues identified by docking were changed and some new residues were introduced in the proximity of ligands. Our results are in line with the majority of previous mutational reports. These results show that hydrophobicity is the determining factor of CCR5 antagonism. In addition, salt bridging and hydrogen bond contacts between ligands (14, 25, and 37) and CCR5 are also crucial for inhibitory activity. The residues newly identified by MD simulation are Ser160, Phe166, Ser180, His181, and Trp190, and so far no site-directed mutagenesis studies have been reported. To determine the contributions made by these residues, additional mutational studies are suggested. We propose a general binding mode for these derivatives based on the MD simulation results of higher (14), medium (37), and lower (25) potent inhibitors. Interestingly, we found some trend for these inhibitors such as, salt bridge interaction between basic nitrogen of ligand and acidic Glu283 seemed necessary for inhibitory activity. Also, two aromatic pockets (pocket I – TM1-3 and pocket II – TM3-6) were linked by the central polar region in TM7, and the simulated inhibitors show important interactions with the Trp86, Tyr89, Tyr108, Phe112, Ile198, Tyr251, Leu255, and Gln280 and Glu283 residues. These results shed light on the usage of MD simulation to identify more stable, optimal binding modes of the inhibitors.     

Intermolecular insights into allosteric inhibition of histone lysine-specific demethylase 1

BackgroundHistone lysine-specific demethylase 1 (LSD1) has become a potential anticancer target for the novel drug discovery. Recent reports have shown that SP2509 and its derivatives strongly inhibit LSD1 as allosteric inhibitors. However, the binding mechanism of these allosteric inhibitors in the allosteric site of LSD1 is not known yet.MethodsThe stability and binding mechanism of allosteric inhibitors in the binding site of LSD1 were evaluated by molecular docking, ligand-based pharmacophore, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM/GBSA) analysis, quantum mechanics/molecular mechanics (QM/MM) calculation and Hirshfeld surface analysis.ResultsThe conformational geometry and the intermolecular interactions of allosteric inhibitors showed high binding affinity towards allosteric site of LSD1 with the neighboring amino acids (Gly358, Cys360, Leu362, Asp375 and Glu379). Meanwhile, MD simulations and MM/GBSA analysis were performed on selected allosteric inhibitors in complex with LSD1 protein, which confirmed the high stability and binding affinity of these inhibitors in the allosteric site of LSD1.ConclusionThe simulation results revealed the crucial factors accounting for allosteric inhibitors of LSD1, including different protein–ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. Meanwhile, a halogen bond interaction between chlorine atom of ligand and key residues Trp531 and His532 was recurrent in our analysis confirming its importance.General significanceOverall, our research analyzed in depth the binding modes of allosteric inhibitors with LSD1 and could provide useful information for the design of novel allosteric inhibitors.     

Molecular dynamics simulations reveal how the reticulon-homology domain of the autophagy receptor RETREG1/FAM134B remodels membranes for efficient selective reticulophagy

ABSTRACT

The autophagy receptor for selective reticulophagy, RETREG1/FAM134B is essential for ER maintenance, and its dysfunction is associated with neuronal disorders, vascular dementia, or viral infections. The protein consists of the reticulon-homology domain (RHD) that is flanked at the N- and C-termini by an intrinsically disordered protein region (IDPR), where the C terminal IDPR carries the indispensable LC3-interacting region (LIR) motif for the interaction with LC3. The RHD of RETREG1 is presumed to play a role in membrane remodeling, but the absence of a known 3D structure of this domain so far prevented researchers from gaining mechanistic insights into how the RETREG1 RHD curves membranes, and thereby facilities reticulophagy. The recent study by Bhaskara et al., which is described in this editor’s corner article, used molecular dynamics (MD) simulations to create a structural model of the RETREG1 RHD. MD simulations along with in vitro liposome remodeling experiments reveal how the RHD domain acts on the ER membrane and, in concert with the C terminal IDPR, executes the function of RETREG1 in selective reticulophagy.

Abbreviations: ER, endoplasmic reticulum; IDPR, intrinsically disordered protein region; LIR, LC3-interacting region; MD, molecular dynamics; RHD, reticulon-homology domain; TM, transmembrane     

Mitogen activated protein kinase-1 and cell division control protein-42 are putative targets for the binding of novel natural lead molecules: a therapeutic intervention against Candida albicans

Abstract

Candida albicans, fungal yeast causes several lethal infections in immune-suppressed patients and recently emerged as drug-resistant pathogens worldwide. The present study aimed to screen putative drug targets of Candia albicans and to study the binding potential of novel natural lead compounds towards these targets by computational virtual screening and molecular dynamic (MD) simulation. Through extensive analysis of mitogen-activated protein kinase (MAPK) signalling pathways, mitogen-activated protein kinase-1 (HOG1) and cell division control protein-42 (CDC42) genes were prioritized as putative targets based on their virulent functions. The three-dimensional structures of these genes, not available in their native forms, were computationally modeled and validated. 76 lead molecules from various natural sources were screened and their drug likeliness and pharmacokinetic features were predicted. Among these ligands, two lead molecules that demonstrated ideal drug-likeliness and pharmacokinetic features were docked against HOG1 and CDC42 and their binding potential was compared with the binding of conventional drug Fluconazole with their usual target. The prediction was computationally validated by MD simulation. The current study revealed that Cudraxanthone-S present in Cudrania cochinchinensis and Scutifoliamide-B present in Piper scutifolium exhibited ideal drug likeliness, pharmacokinetics and binding potential to the prioritized targets in comparison with the binding of Fluconazole and their usual target. MD simulation showed that CDC42-Cudraxanthone-S and HOG1-Scutifoliamide-B complexes were exhibited stability throughout MD simulation. Thus, the study provides significant insight into employing HOG1 and CDC42 of MAPK as putative drug targets of C. albicans and Cudraxanthone-S and Scutifoliamide-B as potential inhibitors for drug discovery.

Communicated by Ramaswamy H. Sarma     

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy

IntroductionMicrochimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues.MethodFluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls.ResultsMicrochimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm² tissue) compared to controls (0.019 ± 0.007 cells/mm² tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm²). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm² versus 0 ± 0/mm²p = 0.003 and 0.043 ± 0.023/mm² versus 0 ± 0/mm²p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype.ConclusionsMicrochimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.     

Homology modeling and examination of the effect of the D92E mutation on the H5N1 nonstructural protein NS1 effector domain

Virulent H5N1 strains of influenza virus often harbor a D92E point mutation in the nonstructural protein NS1. This crucial mutation has been correlated with increased virulence and/or cytokine resistance, but the structural implications of such a change are still unclear. Furthermore, NS1 protein could also be a potential target for the development of novel antiviral agents against H5N1 strains. Therefore, a reasonable 3D model of H5N1 NS1 is important for the understanding of the molecular basis of increased virulence and the design of novel antiviral agents. Based on the crystal structure of a non-H5N1 NS1 protein, a model of H5N1 NS1 was developed by homology modeling, molecular mechanics and molecular dynamics simulations. It was found that the D92E mutation could result in weakened interactions of the carboxylate side chain with other phosphorylated residues, thereby activating phosphorylation of NS1. Figure Superposition of snapshots picked from the two molecular dynamic (MD) trajectories: a H5N1 NS1 homology model and b non-H5N1 NS1 crystal structure after 0 (green ribbon), 5 (blue ribbon) and 10 ns (pink ribbon) MD simulation     

Regiospecificity of Human Cytochrome P450 1A1-Mediated Oxidations: The Role of Steric Effects

Abstract

Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobiotic benzo[a]pyrene (B[a]P) and endogenous fatty acid precursors of prostaglandins, such as arachidonic acid (AA) and eicosapentaenoic acid (EA). We have investigated the extent to which enzyme-substrate interactions govern regio- and stereoselectivity of oxidation of these compounds by using docking and molecular dynamics (MD) simulations to examine the likelihood of substrate oxidation at various sites. Due to structural differences between the substrates analyzed, B[a]P and its diols (planar, rigid), and the fatty acids AA and EA (long, flexible), different docking strategies were required. B[a]P, B[a]P-7,8-diols, (+) 7S,85- and (-) 7R,8R-diols, were docked into the active site of a homology model of P450 1A1 using an automated routine. Affinity (Accelrys, San Diego, CA). AA and EA, on the other hand, required a series of restrained MD simulations to obtain a variety of productive binding modes. All complexes were evaluated by MD-based in silico site scoring to predict product profiles based on certain geometric criteria, such as angle and distance of a given substrate atom from the ferryl oxygen. For all substrates studied, the in vitro profiles were generally reflected by the in silico scores, which suggests that steric factors play a key role in determining regiospecificity in P450 1A1-mediated oxidations. We have also shown that molecular dynamics simulations may be very useful in determination of product profiles for structurally diverse substrates of P450 enzymes.     

Calcium-induced conformational changes of Thrombospondin-1 signature domain: implications for vascular disease

Context: Thrombospondin1 (TSP1) participates in numerous signaling pathways critical for vascular physiology and disease. The conserved signature domain of thrombospondin 1 (TSP1-Sig1) comprises three epidermal growth factor (EGF), 13 calcium-binding type 3 thrombospondin (T3) repeats, and one lectin-like module arranged in a stalk-wire-globe topology. TSP1 is known to be present in both calcium-replete (Holo-) and calcium-depleted (Apo-) state, each with distinct downstream signaling effects.

Objective: To prepare a homology model of TSP1-Sig1 and investigate the effect of calcium on its dynamic structure and interactions.

Methods: A homology model of Holo-TSP1-Sig1 was prepared with TSP2 as template in Swissmodel workspace. The Apo-form of the model was obtained by omitting the bound calcium ions from the homology model. Molecular dynamics (MD) simulation studies (100?ns) were performed on the Holo- and Apo- forms of TSP1 using Gromacs4.6.5.

Results and discussion: After simulation, Holo-TSP1-Sig1 showed significant reorientation at the interface of the EGF1-2 and EGF2-3 modules. The T3 wire is predicted to show the maximum mobility and deviation from the initial model. In Apo-TSP1-Sig1 model, the T3 repeats unfolded and formed coils with predicted increase in flexibility. Apo-TSP1-Sig1model also predicted the exposure of the binding sites for neutrophil elastase, integrin and fibroblast growth factor 2. We present a structural model and hypothesis for the role of TSP1-Sig1 interactions in the development of vascular disorders.

Conclusion: The simulated model of the fully calcium-loaded and calcium-depleted TSP1-Sig1 may enable the development of its interactions as a novel therapeutic target for the treatment of vascular diseases.     

Cross-Scale Numerical Simulations Using Discrete Particle Models

Abstract

We propose a concept for a homogenous computational model in carrying out cross-scale numerical experiments on liquids. The model employs the particle paradigm and comprises three types of simulation techniques: molecular dynamics (MD), dissipative particle dynamics (DPD) and smoothed particle hydrodynamics (SPH). With respect to the definition of the collision operator, this model may work in different hierarchical spatial and time scales as: MD in the atomistic scale, DPD in the mesoscale and SPH in the macroscale. The optimal computational efficiency of the three types of cross-scale experiments are estimated in dependence on: the system size N-where N is the number of particles-and the number of processors P employed for computer simulation. For the three-hierarchical-stage, as embodied in the MD-DPD-SPH model, the efficiency is proportional to N ^8/7 but its dependence on P is different for each of the three types of cross-scale experiments. The problem of matching the different scales is discussed.     

Structure-based rational design of novel hit compounds for pyruvate dehydrogenase multienzyme complex E1 components from Escherichia coli

Pyruvate dehydrogenase multienzyme complex (PDHc) E1 component plays a pivotal role in cellular metabolism to convert the product of glycolysis (pyruvate) to acetyl-CoA, and has been reported as a potential target for anti-microbial and herbicide. In present study, based on the thiamin diphosphate (ThDP) site, four novel hit compounds with high inhibitory activity against the PDHc-E1 from Escherichia coli were firstly designed by using structure-based molecular docking methods. As expected, among four compounds, the compound 3a is the best inhibitor by far, with IC₅₀ value of 6.88 μM against PDHc-E1 from E. coli. To elucidate the interaction mechanism between the active site of PDHc-E1 and its inhibitor, the docking-based molecular dynamics simulation (MD) and MD-based ab initio fragment molecular orbital (FMO) calculations were also further performed. The positive results indicated that all modeling strategies presented in the current study most like to be an encouraging way in design of novel lead compounds with structural diversity for PDHc-E1 in the future.     

Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways

BackgroundNeointimal formation, mediated by the proliferation and migration of vascular smooth muscle cells (VSMCs), is a common pathological basis for atherosclerosis and restenosis. Myricetin, a natural flavonoid, reportedly exerts anti-atherosclerotic effects. However, the effect and mechanism of myricetin on VSMCs proliferation and migration and neointimal hyperplasia (NIH) remain unknown.PurposeWe investigated myricetin's effect on NIH, as well as the potential involvement of transforming growth factor-beta receptor 1 (TGFBR1) signaling in mediating myricetin's anti-atherosclerotic and anti-restenotic actions.MethodsMyricetin's effects on the proliferation and migration of HASMCs and A7R5 cells were determined by CCK-8, EdU assays, wound healing, Transwell assays, and western blotting (WB).Molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance (SPR) and TGFBR1 kinase activity assays were employed to investigate the interaction between myricetin and TGFBR1. An adenovirus vector encoding TGFBR1 was used to verify the effects of myricetin. In vivo, the left common carotid artery (LCCA) ligation mouse model was adopted to determine the impacts of myricetin on neointimal formation and TGFBR1 activation.ResultsMyricetin dose-dependently inhibited the migration and proliferation in VSMCs, suppressed the expression of CDK4, cyclin D3, MMP2, and MMP9. Molecular docking revealed that myricetin binds to key regions for TGFBR1 antagonist binding, and the binding energy was -9.61 kcal/mol. MD simulation indicated stable binding between TGFBR1 and myricetin. Additionally, SPR revealed an equilibrium dissociation constant of 4.35 × 10⁻⁵ M between myricetin and TGFBR1. According to the TGFBR1 kinase activity assay, myricetin directly inhibited TGFBR1 kinase activity (IC₅₀ = 8.551 μM). Furthermore, myricetin suppressed the phosphorylation level of TGFBR1, Smad2, and Smad3 in a dose-dependent pattern, which was partially inhibited by TGFBR1 overexpression. Consistently, TGFBR1 overexpression partially rescued the suppressive roles of myricetin on VSMCs migration and proliferation. Moreover, myricetin dramatically inhibited NIH and reduced TGFBR1, Smad2, and Smad3 phosphorylation in the LCCA.ConclusionThis is the first study to demonstrate that myricetin suppresses NIH and VSMC proliferation and migration via inhibiting TGFBR1 signaling. Myricetin can be developed as a potential therapeutic candidate for treating atherosclerosis and vascular restenosis.     

Vitamin D Intake,Month the Mammogram Was Taken and Mammographic Density in Norwegian Women Aged 50–69

BackgroundThe role of vitamin D in breast cancer etiology is unclear. There is some, but inconsistent, evidence that vitamin D is associated with both breast cancer risk and mammographic density (MD). We evaluated the associations of MD with month the mammogram was taken, and with vitamin D intake, in a population of women from Norway—a country with limited sunlight exposure for a large part of the year.Methods3114 women aged 50–69, who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2004 or 2006/07, completed risk factor and food frequency (FFQ) questionnaires. Dietary and total (dietary plus supplements) vitamin D, calcium and energy intakes were estimated by the FFQ. Month when the mammogram was taken was recorded on the mammogram. Percent MD was assessed using a computer assisted method (Madena, University of Southern California) after digitization of the films. Linear regression models were used to investigate percent MD associations with month the mammogram was taken, and vitamin D and calcium intakes, adjusting for age, body mass index (BMI), study year, estrogen and progestin therapy (EPT), education, parity, calcium intakes and energy intakes.ResultsThere was no statistical significant association between the month the mammogram was taken and percent MD. Overall, there was no association between percent MD and quartiles of total or dietary vitamin D intakes, or of calcium intake. However, analysis restricted to women aged <55 years revealed a suggestive inverse association between total vitamin D intake and percent MD (p for trend = 0.03).ConclusionOverall, we found no strong evidence that month the mammogram was taken was associated with percent MD. We found no inverse association between vitamin D intake and percent MD overall, but observed a suggestive inverse association between dietary vitamin D and MD for women less than 55 years old.     

Identification of Potential Small Molecule Allosteric Modulator Sites on IL-1R1 Ectodomain Using Accelerated Conformational Sampling Method

The interleukin-1 receptor (IL-1R) is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1) ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD) simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.     

Molecular dynamics simulation of proteins under high pressure: Structure,function and thermodynamics

BackgroundMolecular dynamics (MD) simulation is well-recognized as a powerful tool to investigate protein structure, function, and thermodynamics. MD simulation is also used to investigate high pressure effects on proteins. For conducting better MD simulation under high pressure, the main issues to be addressed are: (i) protein force fields and water models were originally developed to reproduce experimental properties obtained at ambient pressure; and (ii) the timescale to observe the pressure effect is often much longer than that of conventional MD simulations.Scope of reviewFirst, we describe recent developments in MD simulation methodologies for studying the high-pressure structure and dynamics of protein molecules. These developments include force fields for proteins and water molecules, and enhanced simulation techniques. Then, we summarize recent studies of MD simulations of proteins in water under high pressure.Major conclusionsRecent MD simulations of proteins in solution under pressure have reproduced various phenomena identified by experiments using high pressure, such as hydration, water penetration, conformational change, helix stabilization, and molecular stiffening.General significanceMD simulations demonstrate differences in the properties of proteins and water molecules between ambient and high-pressure conditions. Comparing the results obtained by MD calculations with those obtained experimentally could reveal the mechanism by which biological molecular machines work well in collaboration with water molecules.     

Full Length Vpu from HIV-1: Combining Molecular Dynamics Simulations with NMR Spectroscopy

Abstract

Based on structures made available by solution NMR, molecular models of the protein Vpu from HIV-1 were built and refined by 6 ns MD simulations in a fully hydrated lipid bilayer. Vpu is an 81 amino acid type I integral membrane protein encoded by the human immunodeficiency virus type-1 (HIV-1) and closely related simian immunodeficiency viruses (SIVs). Its role is to amplify viral release. Upon phosphorylation, the cytoplasmic domain adopts a more compact shape with helices 2 and 3 becoming almost parallel to each other. A loss of helicity for several residues belonging to the helices adjacent to both ends of the loop region containing serines 53 and 57 is observed. A fourth helix, present in one of the NMR-based structures of the cytoplasmic domain and located near the C-terminus, is lost upon phosphorylation.     

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.     

Development of Human Recombinant Antibodies Against ROR1 Tumor Antigen

Background:Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on many types of cancer cells, but not normal adult cells. ROR1 antigen contributes to cancer development and progression by several signaling pathways. ROR1 expression has been associated with tumor growth, survival, and metastasis. In this study specific human recombinant antibodies were selected against ROR1 antigen for their use in cancer immunotherapy.Methods:Phage display technology was used to produce phage antibody from a human scFv library. Phage concentration was determined to confirm the phage rescue process. Panning procedure was performed to isolate specific scFv clones against ROR1 epitope. Phage ELISA was done to evaluate the reactivity of the selected scFvs.Results:Two specific human scFvs with frequencies of 20% and 25% were selected against ROR1 peptide. The antibodies showed specific reaction to the corresponding epitopes in phage ELISA.DiscussionCancer targeted therapy using human specific antibodies is a new strategy, which is used in cancer therapy. The selected specific scFvs that target ROR1 epitope are human antibodies that originated from a human library and have the potential to be used in clinic in cancer immunotherapy of ROR1 positive tumors without induction of human anti mouse antibody (HAMA) response.Key Words: ROR1, Phage display, scFV library, Cancer