Biomedical subjects

Z Shao

Publications and source records attributed to Z Shao.

At least 19 recordsLinked to original sources

PubMed2002

Near field optical microscopy in aqueous solution: implementation and characterization of a vibrating probe.

Near field optical microscopy (NSOM) is one of the possible solutions to circumvent the diffraction limit, but the control of the optical probe in solution has been a technical challenge for practical applications. Most recently, it has been shown that the pipette used in the scanning ion conductance microscope can be modified to form a high resolution near field optical probe. When combined with a novel distance modulation mechanism, a robust near field microscope can be constructed for operation in aqueous solution. In this paper, we present technical details of this design and a further characterization of the NSOM system for imaging in solution. Fundamental limitations of this approach in comparison to other systems are also discussed. Based on the current technology, it is concluded that better than 50 nm resolution should be achievable with this technique for fluorescence, as well as fluorescence resonance energy transfer, imaging of biological specimens.

Microscopy↗

PubMed2001

The effect of spinning conditions on the mechanics of a spider's dragline silk.

We studied the mechanical properties of dragline threads of the edible golden silk spider Nephila edulis that are produced under spinning speeds ranging from 0.1 to 400 mm s(-1) and temperatures ranging from 5 to 40 degrees C. These conditions affected the silk in all of the mechanical traits we tested (strain at breaking, breaking energy, initial Young's modulus and point of yielding). We argue that both trade-offs (between mechanical properties) and constraints (in the manufacturing process) have a large role in defining spider silk fibres.

Animals↗

PubMed2001

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic "hot spot" structure, a subcellular distribution that is characteristic of Tax in HEK cells.

Binding Sites↗

PubMed2001

A20 inhibits NF-kappa B activation downstream of multiple Map3 kinases and interacts with the I kappa B signalosome.

A20, a TNF inducible gene, inhibits TNF-mediated apoptosis as well as NF-kappa B induced by this cytokine. Reporter assay experiments revealed that A20 is a very effective inhibitor of NF-kappa B signaling induced by TRAFs and several Map3 kinases, including NIK, MEKK1, COT, and TAK1. Similarly, the NF-kappa B inducing activity of TAX, an activator of the I kappa B kinase complex, is also abrogated by A20. Inhibition of NF-kappa B is specific as A20 has no effect on TNF-alpha-induced JNK activation. These results suggest that the molecular target of A20 is more distal to the receptor than TRAFs as previously proposed. A20 inhibits NF-kappa B-dependent transcription without a concomitant decrease in nuclear NF-kappa B DNA binding activity or nuclear translocation of p65. This apparent discrepancy between transcriptional readout and gel shift experiments is observed with a variety of stimuli, including expression of IKK beta. Therefore, in addition to the phosphorylation of I kappa B, another signal is needed for transcriptional activation of NF-kappa B. A20 inhibits this non-redundant signal. The observation that A20 associates with IKK alpha and is phosphorylated upon IKK beta co-expression may suggest that A20 interferes with some aspects of signalosome function.

Active Transport, Cell Nucleus↗

PubMed2001

A Drosophila Polycomb group complex includes Zeste and dTAFII proteins.

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20-50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.

Animals↗

PubMed2001

Characterization of a p75(NTR) apoptotic signaling pathway using a novel cellular model.

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.

Apoptosis↗

PubMed2001

Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, cryo-AFM, and cross-linking.

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.

Bacillus Phages↗

PubMed2001

Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein.

We report a novel gene, designated Humanin (HN) cDNA, that suppresses neuronal cell death by K595N/M596L-APP (NL-APP), a mutant causing familial Alzheimer's disease (FAD), termed Swedish mutant. Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP. HN suppressed neurotoxicity by Abeta1-43 in the absence of N2 supplement, but could not inhibit Abeta secretion from NL-APP. HN could also protect neuronal cells from death by NL-APP lacking the 41st and 42nd residues of the Abeta region. Therefore, HN suppressed neuronal cell death by NL-APP not through inhibition of Abeta42 secretion, but with two targets for its inhibitory action: (i) the intracellular toxic mechanism directly triggered by NL-APP and (ii) neurotoxicity by Abeta. HN will contribute to the development of curative therapy of AD, especially as a novel reagent that could mechanistically supplement Abeta-production inhibitors.

Alzheimer Disease↗

PubMed2001

Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein.

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.

Alzheimer Disease↗

PubMed2001

Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy.

The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly defined to the well ordered state was monitored by time-resolved Fourier transform infrared spectroscopy (FTIR) for the first time. From the analysis of FTIR difference spectra, taken on time scales as short as 6 s and up to 1 h after addition of ethanol, intensity vs. time plots of an increasing band at 1618 cm(-1) were observed indicating formation of a beta-sheet coincident with the loss of intensity of a band at 1668 cm(-1) indicating decreases of random coil and/or silk I structure. Both infrared markers were fitted with identical biphasic exponential decay functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is indicated to either proceed sequentially through (at least) two intermediate states that contain different levels of beta-sheet structure or to have parallel pathways of initial beta-sheet formation followed by a slower 'perfection' phase. The first observed process forms in a burst phase a few seconds after mixing (or even faster), prior to the collection of the first spectrum at 6 s. The second observed process occurs with a time constant of approximately 0.5 min, the intermediate present at this stage then continues with a time constant of 5.5 min completing the observed formation of the beta-sheet. The conformation transition of this slower intermediate is not only indicated by an analysis of the kinetics of the random coil and beta-sheet-specific bands discussed above, it roughly coincides with the appearance of an additional infrared marker at 1695 cm(-1), which may be a marker for beta-sheet structure specific to the formation of the perfected structure. The conformation transition of this protein analyzed by infrared spectroscopy provides insight into a part of the fascinating process of cocoon formation in B. mori.

Animals↗

PubMed2001

beta-Amyloid(1-42) peptide directly modulates nicotinic receptors in the rat hippocampal slice.

Alzheimer's disease (AD) is a human neurological disorder characterized by an increasing loss of cognitive function and the presence of extracellular neuritic plaques composed of the beta-amyloid peptide (Abeta(1-42)). However, the link between these molecular correlates of AD and the loss of cognitive function has not been established. The pathology associated with AD includes the loss of basal forebrain cholinergic neurons, presynaptic terminals in the neocortex and hippocampus, and a decrease in the total amount of neuronal nicotinic acetylcholine receptors (nAChRs). This leads to the hypothesis that failure in the cholinergic system underlies the dementia seen in AD. Cognitive performance has been linked to nAChR function in the hippocampus, and the interneurons expressing nAChRs coordinate the activity of large numbers of principal cells and therefore have a powerful role in the regulation of hippocampal activity. We have found that Abeta(1-42) inhibits whole-cell and single-channel nicotinic currents from rat hippocampal interneurons by directly blocking the postsynaptic nAChR channels at concentrations as low as 100 nm. This inhibition appears specific for peptide sequence and neuronal nAChRs, and the magnitude of Abeta(1-42) inhibition is dependent on the nAChR channel subtype expressed. Thus, chronic inhibition of cholinergic signaling by Abeta(1-42) could contribute to the cognitive deficits associated with AD.

Acetylcholine↗

PubMed2001

Role of opioid delta1 receptors, mitochondrial K(ATP) channels, and protein kinase C during cardiocyte apoptosis.

Opioids attenuate cardiac injury after ischemia and reperfusion. We wanted to determine whether the protection of opioids is mediated by blocking cardiocyte apoptosis, and if so, to describe the role of opioid delta1 receptors and protein kinase C (PKC) in this effect. Chick embryonic cardiomyocytes were subjected to 12 h of simulated ischemia and then 12 h of re-oxygenation, which resulted in 54+/-3% (n=6) of cell apoptosis (n=6) as measured by flow cytometry. This result was consistent with DNA laddering and TUNEL assay. Preconditioning, elicited with three cycles of 1 min of simulated ischemia separated by 5 min of reoxygenation before prolonged simulated ischemia, reduced apoptosis (36+/-4%, n=6*). Pretreatment with BNTX (0.1 micromol/l), a selective opioid delta1 receptor blocker, abolished the effects of preconditioning (57+/-5%, n=6). The selective opioid delta receptor agonist BW373U86 (20 pmol/l) also attenuated apoptosis (39+/-3%, n=6* v control). These effects were abolished by 5-hydroxydecanoate (100 microm), a selective mitochondrial K(ATP) channel blocker (50+/-5%, n=6) and by Go-6976 (0.1 micromol/l), a specific PKC inhibitor. Both preconditioning and BW373U86 activated the PKC delta isoform of particulate fraction before simulated ischemia without effect on total and cytosolic fractions. Stimulation of opioid delta1 receptors activates mitochondrial K(ATP) channels and the PKC delta isoform in cultured ventricular myocytes. This is one important signal transduction pathway through which ischemic preconditioning blocks apoptosis and preserves cardiac function.

Adenosine Triphosphate↗

PubMed2001

Characterization of AC mode scanning ion-conductance microscopy.

A scanning ion-conductance microscope (SICM) with a vibrating probe has been recently developed (vSICM). In this system, the amplitude of the AC ionic current is detected by using a lock-in amplifier locked to the vibration frequency of the probe. Such a scheme allows for a better control of the tip position because the AC ionic current is more sensitive to the probe-surface distance than the DC ionic current used previously. In this paper, we demonstrate the utility of this technique to the imaging of topographically rough specimens and high-resolution imaging over selected small areas. We also show that it is possible to record the DC ionic current simultaneously during the scan, which can reveal additional information not apparent in the images obtained with the AC ionic current.

Electric Conductivity↗

PubMed2001

Qian-Kun-Nin, a Chinese herbal medicine formulation, attenuates mitochondrial oxidant stress in cardiomyocytes.

Qian-Kun-Nin is a Chinese herbal medicine formulation used for several indications, including the treatment of cardiovascular diseases. This formulation contains herbs which possess antioxidant properties. In this study, Qian-Kun-Nin's ability to confer protection to cardiomyocytes against reactive oxygen species (ROS) generated during mitochondrial electron transport inhibition was tested. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA, sensitive to H(2)O(2) and hydroxyl radicals) was used to assess intracellular ROS, and propidium iodide (PI) was used to assess viability in cultured chick embryonic cardiomyocytes. Qian-Kun-Nin significantly attenuated oxidation of DCFH in cells exposed to the mitochondrial site III inhibitor, antimycin A, consistent with a decrease in oxidative stress. These attenuated oxidant levels were associated with improved cell survival. After antimycin A exposure, Qian-Kun-Nin decreased cell death from 51. 6+/-3.3% in untreated cells to 27.3+/-3.8% in treated cells at 2 h. We conclude that Qian-Kun-Nin attenuates oxidant stress and protects cells from lethal oxidant damage during mitochondrial electron transport inhibition, and thus its therapeutic potential in treating cardiovascular diseases may relate to its antioxidant properties.

Analysis of Variance↗

PubMed2001

Reconstitution of a functional core polycomb repressive complex.

The opposing actions of polycomb (PcG) and trithorax group (trxG) gene products maintain essential gene expression patterns during Drosophila development. PcG proteins are thought to establish repressive chromatin structures, but the mechanisms by which this occurs are not known. Polycomb repressive complex 1 (PRC1) contains several PcG proteins and inhibits chromatin remodeling by trxG-related SWI/SNF complexes. We have defined a functional core of PRC1 by reconstituting a stable complex using four recombinant PcG proteins. One subunit, PSC, can also inhibit chromatin remodeling on its own. These PcG proteins create a chromatin structure that has normal nucleosome organization and is accessible to nucleases but excludes hSWI/SNF.

Animals↗

PubMed2001

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.

Animals↗

PubMed2001

Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution.

The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 +/- 1 nm, whereas the smaller inter-filament distance, 8 +/- 1 nm, was consistent with interdigitation of the filaments. The 10 +/- 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.

Actin Cytoskeleton↗

PubMed2001

Improved precision of quantitating calcium in biological electron probe analysis.

We describe the use of direct multiple least-squares fitting of (unfiltered) biological X-ray spectra. Test of this method on computer-generated spectra and experimental specimens shows that, for the average biological specimen, direct fitting improves the precision of Ca measurements by an approximate factor of 1.6 for a given electron dose. The influence of non-statistical, instrumental sources of measurement errors is considered, and we show a simple method for reducing the effect of these sources of error in the directly fitted spectra.

Animals↗