Biomedical subjects

Huilin Li

Publications and source records attributed to Huilin Li.

28 records · Page 2Linked to original sources

PubMed2004

Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles.

Previous studies have documented the expression of four kinetically distinct voltage-gated K(+) (Kv) currents, I(to,f), I(to,s), I(K,slow) and I(ss), in mouse ventricular myocytes and demonstrated that I(to,f) and I(to,s) are differentially expressed in the left ventricular apex and the interventricular septum. The experiments here were undertaken to test the hypothesis that there are further regional differences in the expression of Kv currents or the Kv subunits (Kv4.2, Kv4.3, KChIP2, Kv1.5, Kv2.1) encoding these currents in adult male and female (C57BL6) mouse ventricles. Whole-cell voltage-clamp recordings revealed that mean (+/-s.e.m.) peak outward K(+) current and I(to,f) densities are significantly (P < 0.001) higher in cells isolated from the right (RV) than the left (LV) ventricles. Within the LV, peak outward K(+) current and I(to,f) densities are significantly (P < 0.05) higher in cells from the apex than the base. In addition, I(to,f) and I(K,slow) densities are lower in cells isolated from the endocardial (Endo) than the epicardial (Epi) surface of the LV wall. Importantly, similar to LV apex cells, I(to,s) is not detected in RV, LV base, LV Epi or LV Endo myocytes. No measurable differences in K(+) current densities or properties are evident in RV or LV cells from adult male and female mice, although I(to,f), I(to,s), I(K,slow) and I(ss) densities are significantly (P < 0.01) higher, and action potential durations at 50% (APD(50)) are significantly (P < 0.05) shorter in male septum cells. Western blot analysis revealed that the expression levels of Kv4.2, Kv4.3, KChIP2, Kv1.5 and Kv2.1 are similar in male and female ventricles. In addition, consistent with the similarities in repolarizing Kv current densities, no measurable differences in ECG parameters, including corrected QT (QT(c)) intervals, are detected in telemetric recordings from adult male and female (C57BL6) mice.

Animals↗

PubMed2004

Visualizing the RNA molecule in the bacterially expressed vesicular stomatitis virus nucleoprotein-RNA complex.

Packaging of the RNA molecule in viruses is important for the preservation and expression of viral genomic information. The vesicular stomatitis virus (VSV) nucleoproteins are kept associated with its negative-strand RNA during the mRNA synthesis and replication, in contrast to the tobacco mosaic virus whose nucleoproteins are released from RNA. It has been a puzzle how the VSV RNA is packaged to meet the contradicting requirements of protection and the accessibility to the polymerase. We report an 18 A resolution structure of the recombinant nucleoprotein-RNA complex determined by single-particle electron microscopy. In the 3D density map, a ring of density is resolved on the inner surface and the density is proposed to be the RNA. The RNA is located on the inner surface of the decameric complex near the top end. This is dramatically different from the RNA packaging in TMV, but consistent with previously published biochemical findings.

Amino Acid Sequence↗

PubMed2003

Self-assembly in aqueous solution of wheel-shaped Mo154 oxide clusters into vesicles.

Surfactants and membrane lipids readily assemble into complex structures such as micelles, liposomes or hollow vesicles owing to their amphiphilic character-the fact that part of their structure is attracted to polar environments while another part is attracted to non-polar environments. The self-assembly of complex structures also occurs in polyoxometallate chemistry, as exemplified by the molybdenum blue solutions known for centuries. But while the presence of nanometre-sized metal oxide aggregates in these solutions has long been recognized, unravelling the composition and formation process of these aggregates proved difficult. Recent work has indicated that discrete, wheel-shaped mixed-valence polyoxomolybdate clusters of the type [Mo154] (refs 2-4) assemble into well-defined nanometre-sized aggregates, including spherical structures. Here we report light-scattering data and transmission electron microscopy images of hollow spherical structures with an average, almost monodisperse radius of about 45 nm and composed of approximately 1,165 [Mo154] wheel-shaped clusters. The clusters appear to lie flat and homogeneously distributed on the vesicle surface. Unlike conventional lipid vesicles, the structures we observe are not stabilized by hydrophobic interactions. Instead, we believe the polyoxomolybdate-based vesicles form owing to a subtle interplay between short-range van der Waals attraction and long-range electrostatic repulsion, with important further stabilization arising from hydrogen bonding involving water molecules encapsulated between the wheel-shaped clusters and in the vesicles' interior.

Journal Article↗

PubMed2003

Selective elimination of I(K,slow1) in mouse ventricular myocytes expressing a dominant negative Kv1.5alpha subunit.

Although previous studies have revealed a role for the voltage-gated K+ channel alpha-subunit Kv1.5 (KCNA5) in the generation of the 4-aminopyridine (4-AP)-sensitive component of delayed rectification in mouse ventricles (IK,slow1), the phenotypic consequences of manipulating IK,slow1 expression in vivo in different (mouse) models are distinct. In these experiments, point mutations were introduced in the pore region of Kv1.5 to change the tryptophan (W) at position 461 to phenylalanine (F) to produce a nonconducting subunit, Kv1.5W461F, that is shown to function as a Kv1 subfamily-specific dominant negative (Kv1.5DN). With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, three lines of Kv1.5DN-expressing (C57BL6) transgenic mice were generated and characterized. Electrophysiological recordings from Kv1.5-DN-expressing left ventricular myocytes revealed that the micromolar 4-AP sensitive IK,slow1 is selectively eliminated. The attenuation of IK,slow1 is accompanied by increased ventricular action potential durations and marked QT prolongation. In contrast to previous findings in mice expressing a truncated (DN) Kv1.1 transgene; however, no electrical remodeling is evident in Kv1.5DN-expressing ventricular myocytes, and the (Kv1.5DN-induced) elimination of IK,slow1 does not result in spontaneous ventricular arrhythmias.

Action Potentials↗

PubMed2003

Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle.

Expression of the transient outward K+ current (Ito) was analysed in nine different regions of the canine ventricle. In addition to the previously described transmural gradients in the right and left ventricular free walls, an inverted U-shaped pattern of Ito expression was observed in the interventricular septum. The mRNA and protein expression for the K+ channel beta subunit KChIP2 were examined in the same tissues. The KChIP2 protein levels closely matched mRNA expression in all regions of the ventricle and paralleled the density of Ito. The global pattern of gene expression in human epicardial and endocardial tissue was examined using microarrays. Only 0.1 % of the genes expressed in the human ventricle displayed the same expression pattern as the KChIP2 gene in left ventricle. It is unlikely, therefore, that the reported distribution of KChIP2 protein within the ventricle can be explained by a cross-reaction of the anti-KChIP2 antibody with a different protein. It is concluded that transcriptional regulation of the KChIP2 gene is a primary determinant of Ito expression in heart.

Animals↗

PubMed2002

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization.

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.

Affinity Labels↗

PubMed2002

Role of heteromultimers in the generation of myocardial transient outward K+ currents.

Previous studies have demonstrated a role for Kv4 alpha subunits in the generation of the fast transient outward K+ current, I(to,f), in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular I(to,f). Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in I(to,f) density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate I(to,f) in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K+ currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native I(to,f) than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular I(to,f) channels are heteromeric, comprising Kv4.2/Kv4.3 alpha subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in I(to,f) expression in adult mouse ventricle.

Animals↗

PubMed2002

Microtubule structure at 8 A resolution.

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined.

Cryoelectron Microscopy↗

PubMed2001

Accurate Recording and Measurement of Electron Diffraction Data in Structural and Difference Fourier Studies of Proteins.

Many of the techniques that have been developed in X-ray crystallography are being applied in electron crystallographic studies of proteins. Electron crystallography has the advantage of measuring structure factor phases directly from high resolution images with an accuracy substantially higher than is common in X-ray crystallography. However, electron diffraction amplitudes are often not as precise as those obtained in X-ray work. We discuss here some approaches to maximizing the reliability of the diffraction amplitudes through choice of exposure and data processing schemes. With accurate measurement of diffraction data, Fourier difference methods can be used in electron crystallographic studies of small, localized changes of proteins that exist in two-dimensional crystals. The mathematical basis for the power of these methods in detecting small changes is reviewed. We then discuss several issues related to optimizing the quality of the diffraction data and derive an expression for the best exposure for recording diffraction patterns. An application of Fourier difference maps in localizing drug binding sites on the protein tubulin is discussed.

Journal Article↗

PubMedDate not supplied

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review).

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.

Adenosine Triphosphate↗