Delineating the Site of Interaction of the 5-HT3A Receptor with the Chaperone Protein RIC-3

The serotonin type 3A (5-HT_3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains: extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Previously, we have demonstrated that the 5-HT_3A-ICD is required for the interaction between 5-HT_3A and the chaperone protein resistance to inhibitors of choline esterase (RIC-3). Additionally, we have shown that 5-HT_3A-ICD fused to maltose-binding protein (MBP) directly interacts with RIC-3, without the involvement of other protein(s). To elucidate the molecular determinants of this interaction, we developed different MBP-fused 5-HT_3A-ICD constructs by deleting large segments of its amino acid sequence. We expressed seven engineered ICDs in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction between MBP-5HT_3A-ICD constructs and RIC-3 was investigated. In summary, we identify a 24-amino-acid-long segment of the 5-HT_3A-ICD as a molecular determinant for the interaction between the 5-HT_3A-ICD and RIC-3.     

Cellular Effects of Curcumin on Plasmodium falciparum Include Disruption of Microtubules

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC₅₀ of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.     

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease^1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically¹². "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution¹². In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) <3000 Pascal and stiff substrata/tissues as those with E >20,000 Pascal.Download video file.^{(114M, mp4)}     

苜蓿中华根瘤菌nifA基因突变影响多种细胞学过程

苜蓿中华根瘤菌nifA基因在共生固氮过程中担负着调控功能,nifA突变株Rm1354在宿主植物的根部诱导白色无效根瘤。本文报道Rm1354在自生状态下的表型变化。nifA的突变导致根瘤菌在半固体培养基上泳动变慢,胞外蛋白含量降低。有趣的是,Rm1354在延宕期间高丝氨酸内酯含量比野生型低,在指数期和静止期却比野生型高。另外,突变株Rm1354的竞争结瘤能力也大大减弱。这些结果揭示了苜蓿中华根瘤菌nifA基因对许多细胞学过程都有调控作用。     

螺类与着生藻类的相互作用及其对沉水植物的影响

浅水湖泊的富营养化常导致水生植被的退化与浮游藻类的爆发［１０,１８,２９］。可利用光通常是决定沉水植物分布、生物量和生产力的最重要因子,因此,伴随高营养负荷的浮游藻类繁殖,极大地削弱了沉水植物的光合能力［２０］。然而,Ｐｈｉｌｉｐｓ等人［２８］认为,...     

Interaction of R17 Coat Protein With Its RNA Binding Site For Translational Repression

Abstract

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kj at 2°C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of K_a and a ΔH = ?19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RN As failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific.

We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.     

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIP_L and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIP_L, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIP_L. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIP_L and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIP_L.     

Comparison of Effects of Inhibitors of Viral and Cellular Protein Kinases on Human Cytomegalovirus Disruption of Nuclear Lamina and Nuclear Egress

Human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 and cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption but did reduce the number of cytoplasmic capsids more than the number of nuclear capsids.     

Isolation and Immunochemical Study of a Soluble Cerebellar Lectin Delineating Its Structure and Function

A procedure of sequential extractions of cerebellar tissue was set up, which allowed specific solubilization of endogenous lectins by mannose. Two cerebellar soluble lectins, CSL1 (Mr = 33,000) and CSL2 (Mr = 31,500), were isolated. They appeared to consist of structurally and immunologically related polypeptides chains. By immunoaffinity, another minor component (Mr = 45,000) was isolated. Immunological studies suggested that the minor component is the precursor of the two other, i.e., CSL1 and CSL2, subunits. CSL1 (mainly lysosomal) possesses an additional peptide compared with CSL2 (mainly cytoplasmic and extracellular), which seems to be implicated in the signal for secretion and release.     

丙二醛对大鼠海马神经元结构的破坏和钙离子稳态的影响

脂质过氧化中间产物丙二醛(Malondialdehyde,MDA)在生物体内表现了广泛的生物毒性,MDA也是机体过度训练和运动性疲劳的重要生理指标.采用光学显微镜和透射式电子显微镜观察不同浓度MDA作用后海马神经元形态和超微结构的变化,并采用荧光分光光度法测定原代培养的海马神经元中Ca2+-ATPase活性的变化和胞质游离钙离子水平的变化,探讨MDA对海马神经元形态和结构上的破坏及神经元钙离子稳态的影响.在光镜下可观察到MDA作用下神经元突触变短,胞体肿胀,出现细胞死亡或凋亡的形态特征;在电镜下可观察到线粒体结构的明显破坏,内膜上的嵴颗粒减少或消失;同时MDA还通过抑制质膜Ca2+-ATPase的活性和其它的途径,破坏神经元胞质游离Ca2+稳态.结果表明,MDA可通过破坏海马神经元的结构和影响胞质中钙离子稳态,破坏神经元的生理功能,在机体运动性中枢疲劳形成中可能发挥重要作用.     

鄂尔多斯乌兰木伦遗址石制品拼合研究及其对遗址成因的指示意义

乌兰木伦遗址是继上世纪20年代发现萨拉乌苏和水洞沟遗址以来,在鄂尔多斯高原时隔近90年新发现的重要旧石器遗址。自2010年发现以来,该遗址已进行了5个年度的连续性发掘,发现了丰富的石制品、动物化石以及用火遗迹等。光释光和碳14年代测定表明古人类在该遗址活动的时间为距今5~6.5万年。本文对乌兰木伦遗址1号地点2010-2012年发掘出土的11934件石制品进行拼合研究,辨别出28个拼合组,含64件石制品,拼合率约为0.53%;其拼合形式可分为拼对(conjoin)和拼接(join)两种类型。石制品拼合组空间分布等相关信息显示,乌兰木伦旧石器时代中期遗址为原地埋藏。     

Integration and Disruption Effects of Shape and Texture in Haptic Search

In a search task, where one has to search for the presence of a target among distractors, the target is sometimes easily found, whereas in other searches it is much harder to find. The performance in a search task is influenced by the identity of the target, the identity of the distractors and the differences between the two. In this study, these factors were manipulated by varying the target and distractors in shape (cube or sphere) and roughness (rough or smooth) in a haptic search task. Participants had to grasp a bundle of items and determine as fast as possible whether a predefined target was present or not. It was found that roughness and edges were relatively salient features and the search for the presence of these features was faster than for their absence. If the task was easy, the addition of these features could also disrupt performance, even if they were irrelevant for the search task. Another important finding was that the search for a target that differed in two properties from the distractors was faster than a task with only a single property difference, although this was only found if the two target properties were non-salient. This means that shape and texture can be effectively integrated. Finally, it was found that edges are more beneficial to a search task than disrupting, whereas for roughness this was the other way round.     

Delineating the roles of males and females in sperm competition

Disentangling the relative roles of males, females and their interactive effects on competitive fertilization success remains a challenge in sperm competition. In this study, we apply a novel experimental framework to an ideally suited externally fertilizing model system in order to delineate these roles. We focus on the chinook salmon, Oncorhynchus tshawytscha, a species in which ovarian fluid (OF) has been implicated as a potential arbiter of cryptic female choice for genetically compatible mates. We evaluated this predicted sexually selected function of OF using a series of factorial competitive fertilization trials. Our design involved a series of 10 factorial crosses, each involving two ‘focal’ rival males whose sperm competed against those from a single ‘standardized’ (non-focal) rival for a genetically uniform set of eggs in the presence of OF from two focal females. This design enabled us to attribute variation in competitive fertilization success among focal males, females (OF) and their interacting effects, while controlling for variation attributable to differences in the sperm competitive ability of rival males, and male-by-female genotypic interactions. Using this experimental framework, we found that variation in sperm competitiveness could be attributed exclusively to differences in the sperm competitive ability of focal males, a conclusion supported by subsequent analyses revealing that variation in sperm swimming velocity predicts paternity success. Together, these findings provide evidence that variation in paternity success can be attributed to intrinsic differences in the sperm competitive ability of rival males, and reveal that sperm swimming velocity is a key target of sexual selection.     

Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.     

酵母双杂交系统在膜蛋白相互作用研究中的应用

膜相关蛋白约占细胞总蛋白质中的1/3,它们大都参与了细胞的诸多生理、病理过程和药物反应机理。研究膜蛋白的相互作用对于揭示细胞的生命活动规律及寻找药物作用靶标都有重要的意义。由于膜蛋白本身的特性及其难以进入核内等原因,经典的酵母双杂交技术并不适用于检测膜蛋白间的相互作用。针对在活细胞中研究膜蛋白相互作用的需要,近年来国际上先后发展了一系列用于膜蛋白相互作用研究的酵母双杂交新系统,并取得了许多重要发现。