Computational identification and analysis of noncoding RNAs - Unearthing the buried treasures in the genorne

The central dogma of molecular biology states that the genetic information flows from DNA to RNA to protein. This dogma has exerted a substantial influence on our understanding of the genetic activities in the cells. Under this influence, the prevailing assumption until the recent past was that genes are basically repositories for protein coding information, and proteins are responsible for most of the important biological functions in all cells. In the meanwhile, the importance of RNAs has remained rather obscure, and RNA was mainly viewed as a passive intermediary that bridges the gap between DNA and protein. Except for classic examples such as tRNAs (transfer RNAs) and rRNAs (ribosomal RNAs), functional noncoding RNAs were considered to be rare     

Calnexin associates with Shaker K+ channel protein but is not involved in quality control of subunit folding or assembly.

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacted transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. We also investigated whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. Our results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels.     

Comparison of modulation of Kv1.3 channel by two receptor tyrosine kinases in olfactory bulb neurons of rodents

Activation of the receptor tyrosine kinase (RTK), insulin (IRK) or neurotrophin B (TrkB), was characterized and compared in olfactory bulb neuron (OBN) cultures from Sprague Dawley rats and sv129 B6 mice. Current suppression attributed to modulation of the delayed rectifier, Kv1.3, a voltage-gated potassium (Kv) channel of the Shaker family, was observed following acute application of the growth factors, insulin or brain-derived neurotrophic factor (BDNF), to mitral cells of either rodent model. Using site-directed mutagenesis of putative tyrosine phosphorylation recognition motifs in the channel, we find that stimulation of Kv1.3 with these growth factors causes multiple phosphorylation, albeit via different residue combinations that are RTK specific.     

A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase.

The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.     

Phylogenomic analysis and evolution of the potassium channel gene family

Potassium channels govern the permeability of cells to potassium ions, thereby controlling the membrane potential. In metazoa, potassium channels are encoded by a large, diverse gene family. Previous analyses of this gene family have focused on its diversity in mammals. Here we have pursued a more comprehensive study in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes. The investigation revealed 164 potassium channel encoding genes in C. elegans, D. melanogaster, and mammals, classified into seven conserved families, which we applied to phylogenetic analysis. The trees are discussed in relation to the assignment of orthologous relationships between genes and vertebrate genome duplication.     

Characterization of human Kv4.2 mediating a rapidly-inactivating transient voltage-sensitive K+ current.

A human cDNA for the voltage-sensitive potassium channel subunit Kv4.2 has been cloned and functionally characterized. The human Kv4.2 (KCND2) gene was mapped at 7q31-32. Kv4.2 mRNA is prominently expressed in human brain. Relatively high concentrations of Kv4.2 mRNA occurred in mRNA preparations of amygdala, caudate nucleus, cerebellum, hippocampus, substantia nigra, and thalamus. Kv4.2 mRNA was not detected in human heart, kidney, liver, lung, pancreas, and skeletal muscle. The derived Kv4.2 open reading frame consists of 630 amino acids. In comparison to rat Kv4.2, the human Kv4.2 sequence is highly conserved showing amino acid sequence differences at five positions only. The Kv4.2 subunits were expressed heterologously in human embryonic kidney (293) cells and mediated a rapidly inactivating, A-type outward K+ current. The gating kinetics of the Kv4.2-mediated currents were very similar to those of rat Kv4.2-mediated currents. Both the Kv4.2 and Kv4.3 subunits have been implicated in mediating the transient outward K+ current Ito in rodent cardiac myocytes. In contrast we did not detect Kv4.2. but solely Kv4.3 mRNA in human heart RNA preparations. This may suggest that Kv4.2 subunits do not contribute to the rapid transient outward K+ current of atrial and ventricular myocytes in humans.     

Stable expression and characterization of human PN1 and PN3 sodium channels

Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.     

Microtexture and Chitin/Calcite Orientation Relationship in the Mineralized Exoskeleton of the American Lobster

The exoskeleton of the American lobster Homarus americanus is a hierarchical nanocomposite consisting of chitin–protein fibers, reinforced with amorphous calcium carbonate (ACC) and a small amount of crystalline calcite. Crystallographic pole‐figure analysis reveals two texture components of the crystalline α‐chitin in the exoskeleton. One component represents the well‐known twisted plywood structure of chitin–protein fibers within the cuticle plane, and the second component represents fibers oriented roughly perpendicular to the cuticle surface. These perpendicular fibers interpenetrate the open canals of the planar honeycomblike structure originating from the well‐developed pore‐canal system present in this material. The calcite crystallites reveal fiber texture with the crystallographic c‐axis oriented perpendicular to the cuticle surface, suggesting an orientation relationship between calcite and the organic chitin–protein fibers. Local orientation analysis using X‐ray microdiffraction reveals that the crystalline calcium carbonate fraction is associated with the chitin–protein fibers oriented perpendicular to the surface. Calcite is exclusively found in the exocuticle and is mostly restricted to a thin layer in the outermost region, while the major part of the exocuticle and the whole endocuticle contain ACC exclusively. It is therefore speculated that the most likely function of calcite in the exoskeleton of the American lobster is related to impact‐ and wear‐resistance.     

Gene amplification and rearrangement of murine ribosomal RNA in virus-induced Rauscher leukemia

Using as a hybridization probe cDNA 35s RNA from the Rauscher leukemia cells, a part of rRNA gene cluster from the gene library of mice C-1 erythroleukemia cells has been cloned. Fragment 6.7 kb recloned into pUC19 rRNA was used as a probe to analyse organization of rRNA genes of mice with RL. The observed amplification and rearrangement in genome DNA of spleen cells are determined by a new type of their rRNA genes rearrangement in the nontranscribed as well as in the transcribed part of rRNA.     

反射表面微变形的改进型龙虾眼透镜

龙虾眼透镜作为一种特殊的透射式反射聚焦元件,在X射线等高能领域具有重要的应用价值,并在可见光、红外波段具有潜在的应用前景。为提高龙虾眼透镜的成像质量,从理论方面分析了变周期闪耀光栅的衍射干涉因子以及衍射场情况,利用变周期闪耀光栅对龙虾眼通道内壁进行微整形,并且对经过微整形之后的一维龙虾眼结构进行了聚焦光场分布计算。通过与之前结构的仿真对比,结果表明:焦距为100 mm时,有效半口径为95 mm的现有结构在焦面处的弥散斑直径由10 mm减小至1 mm,光能集中度由75%增大至89.62%,并且在不同入射高度下焦平面处的光斑直径以及光能集中度均有提高。     

Agonist-like activity of antibodies directed against the second extracellular loop of the human cardiac serotonin 5-HT4(e) receptor in transfected COS-7 cells

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.     

Induction of baboon herpes virus antigens in producing lymphoblastoid cells

Treatment of lymphoid 594S/F9 cells with 12-o-tetradecanoylphorbol-13-acetate (TPA), cycloheximide (CH) has no effect on cell proliferation. A reversible blocking of the cellular DNA and RNA synthesis is observed. Repeated treatment with TPA, iododeoxyuridine (IDU) or sodium butyrate (SB) causes more pronounced changes in cell metabolism, which sharply decreases the salt sensitive DNA polymerase activity. Simultaneously the activity of the virus-induced ammonium sulphate resistant DNA polymerase increases. The combined effect of TPA and CH stimulates the synthesis of viral antigens in certain latently infected cells. The number of productively infected cells remains approximately at the same level for 96 h. IDU has a highest effect on viral antigens expression at repeated induction.     

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.     

Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells.

Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-ICL) were constructed and expressed in Saccharomyces cerevisiae cells. We observed subcellular localization of expressed products of the mutant CT-ICL genes by immunoelectron microscopy. An unknown structure termed a protein aggregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa et al. (1996) J. Electron Microsc. 45: 491-497). We chose two typical cells harbouring the mutant ICL genes delta 550 and delta 237-339 to analyse the ultrastructure and three-dimensional (3D) structure of PABs. The PABs had a homogeneous matrix with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PABs could not be separated from the cytoplasm or mitochondria under a confocal fluorescence microscope, 3D reconstruction of serial electron micrographs clearly showed the PAB was an independent structure of varying size and had the shape of an incomplete sphere. A cell was sometimes observed to have multiple PABs.     

Evidence for voltage-gated potassium channel beta-subunits with oxidoreductase motifs in human and rodent pancreatic beta cells

Voltage-gated K(+) channel alpha subunits (K(V) alpha) have been previously identified in pancreatic islet beta-cells where it has been suggested they have a role in membrane repolarization and insulin secretion. Here we report the cloning of the three mammalian K(V) beta subunits, including splice variants of these subunits, from both human and rat pancreatic islets and from the rat insulinoma cell line INS-1. Two of the splice variants, K(V) beta1a and K(V) beta3, previously reported to be neuronal tissue specific, are expressed in islets and INS-1 cells. In addition, a splice variant of K(V) beta2 that lacks two potential protein kinase C phosphorylation sites at the amino terminus is present. Immunoblot analysis suggests a high level of K(V) beta2 subunit protein in rat pancreatic islets and immunoprecipitation with anti-K(V) beta2 antibody pulls down a protein from INS-1 cells that reacts with anti-aldose reductase antibody. The K(V) beta subunits, which are attached to the cytoplasmic face of the alpha subunits and are members of the aldose reductase superfamily of NADPH oxidoreductases, may have an as yet undetermined role in the regulation of insulin secretion by the intracellular redox potential. Finally, we suggest that a systematic nomenclature for K(V) beta subunits first proposed by McCormack et al. be adopted for this family of potassium channel subunits as it corresponds with the nomenclature used for their cognate K(V) alpha subunits.     

Modeling conserved structure patterns for functional noncoding RNA

RNA regulation has been increasingly recognized as a potential and perhaps overlooked genetics of higher organisms. Noncoding RNAs (ncRNA) may play various catalytic and regulatory roles in the genetic operating system. Recent studies using comparative genomics and molecular genetics show evidence of the presence of varied ncRNAs. Unlike protein coding genes, there is a lack of comparable information or outstanding signal for ncRNAs. Traditional computational linguistics show limitations in modeling complicated secondary structures and prevent us from identifying structure-function relationships of ncRNAs. This paper presents a novel approach, based on a set of distance constraints, to model the predicted RNA secondary structures. Further, a filtering schema is presented to identify matched models for the queried secondary structures.     

毛细管电泳芯片的模拟设计与实验研究

利用ANSYS软件对毛细管电泳芯片微沟道内样品流动情况进行模拟 ,获得了不同进样模式下微沟道的结构与流体流速之间的关系 ,对芯片整体结构参数进行设计 :毛细管微沟道最终尺寸为宽度 16 μm ,深度 10 μm ,有效分离长度为 3.5cm的圆角转弯形沟道。采用激光诱导荧光原理进行实验测试 ,建立测试系统 ,对两段不同长度的DNA片段实现了基线分离 ,研究结果为毛细管电泳芯片的进一步应用奠定了基础     

CDMA cellular engineering issues

The frequency reuse efficiency for the proposed code division multiple access (CDMA) cellular system is analytically derived, and it is shown that the cell capacity of a fully loaded multiple cell system is about 75% of what would be available for a single cell system. In addition, an engineering issue in transitioning from analog advanced mobile phone system (AMPS) to CDMA system is discussed. Calculating excessive interference power due to a CDMA channel, it is shown that when a CDMA channel is introduced in a cell, there should be at least one ring of buffer cells, in which the analog channels falling into the CDMA band are not reused