Biomedical subjects

Z Shao

Publications and source records attributed to Z Shao.

At least 19 recordsLinked to original sources

PubMed2000

A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery.

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Bacteriophages↗

PubMed2000

Cryoatomic force microscopy of filamentous actin.

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of approximately 38 nm. Even at a moderate concentration ( approximately 10 microg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of approximately 13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.

Actin Cytoskeleton↗

PubMed2000

Optimizing the window size for multiple least squares fitting of electron energy loss spectra.

The purpose of this study was to determine the effect of the fitting window size used with the linear least squares fit method when quantitating trace elements with electron energy loss spectroscopy. Theory and computer simulation with a simple model of two 'signals' show that, when the background underlying the signal is slowly varying and the signal is localized, there exists a minimum window optimal for fitting raw spectra. The width of the minimum fitting window can be determined directly from the reference standard spectrum of the signal alone and the estimates of signal and background are related. Use of narrower than the minimum window will increase the fitting uncertainty of the signal and yield less reliable results. More complicated experimental spectra must be fitted to more than two standards and a simple analytical expression of the minimum fitting window cannot be derived, but can be determined empirically. Our study shows that the empirical value obtained from experimental spectra is only slightly larger than the theoretical value derived from the simple model, indicating that this conclusion is still valid. When fitting difference spectra with a slowly varying background, the estimates of signal and background are independent and windows wider than the size of the signal will yield the same fitting uncertainty. In the presence of a non-slowly varying background, common in difference spectra, the minimum window size depends on the fine structure of the signal and the background.

Calcium↗

PubMed2000

PNA-dependent gene chemistry: stable coupling of peptides and oligonucleotides to plasmid DNA.

Two approaches are described for stably conjugating peptides, proteins and oligonucleotides onto plasmid DNA. Both methods use a peptide nucleic acid (PNA) clamp, which binds irreversibly and specifically to a binding site cloned into the plasmid. The first approach uses a biotin-conjugated PNA clamp that can be used to introduce functional biotin groups onto the plasmid to which streptavidin can bind. Atomic force microscopy images of linearized plasmid show streptavidin localized at the predicted PNA binding site on the DNA strand. Peptides and oligonucleotides containing free thiol groups were conjugated to maleimide streptavidin, and these streptavidin conjugates were bound to the biotin-PNA-labeled plasmid. In this way, peptides and oligonucleotides could be brought into stable association with the plasmid. A second approach used a maleimide-conjugated PNA clamp. Methods are described for conjugating thiolated peptides and oligonucleotides directly to the maleimide-PNA-DNA hybrid. This straightforward technology offers an easy approach to introduce functional groups onto plasmid DNA without disturbing its transcriptional activity.

Animals↗

PubMed1999

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

Amino Acid Sequence↗

PubMed1999

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Cloning, Molecular↗

PubMed1999

Images of oligomeric Kv beta 2, a modulatory subunit of potassium channels.

The Shaker type voltage-gated potassium (K+) channel consists of four pore-forming Kv alpha subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kv beta subunits via formation of heteromultimeric Kv alpha-Kv beta complexes. Because each (Kv alpha)4 could recruit more than one Kv beta subunit and different Kv beta subunits could potentially interact, the stoichiometry of alpha-beta and beta-beta complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kv beta 2 subunit in Sf9 insect cells. The purified Kv beta 2, examined by atomic force and electron microscopy techniques, is found predominately as a square-shaped tetrameric complex with side dimensions of 100 x 100 A2 and height of 51 A. Thus, Kv beta 2 is capable of forming a tetramer in the absence of pore-forming alpha subunits. The center of the Kv beta 2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.

Amino Acid Sequence↗

PubMed1999

Stabilization of chromatin structure by PRC1, a Polycomb complex.

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.

Animals↗

PubMed1999

VacA from Helicobacter pylori: a hexameric chloride channel.

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self-associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (P(Cl):P(Na) = 4.2:1), inhibition by the blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and a permeability sequence, SCN- >> I- > Br- > Cl- > F, consistent with a 'weak field strength' binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s(-1)) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane-bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH.

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH approximately 5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.

Bacterial Proteins↗

PubMed1999

A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Animals↗

PubMed1999

Cyclin D1 associates with the TBP-associated factor TAF(II)250 to regulate Sp1-mediated transcription.

We have previously shown that Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. Here we investigated the mechanism(s) through which cyclin D1 represses Sp1. We examined the ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s). The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. We further demonstrate that TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and that recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduced the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) was sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250.

3T3 Cells↗

PubMed1999

Parallel electron energy-loss spectroscopy free from gain variation.

A new method is presented for removing the effect of the gain variation of parallel detectors used for the quantitation of trace elements with electron energy loss spectroscopy (EELS). Use of the ratio of two first-difference spectra eliminates the effect of gain variation of the detector, therefore eliminating the need for gain normalization and dark current subtraction. This method is particularly suitable for revealing small signals superimposed on a large background, a typical scenario for trace element quantitation of both biological and inorganic materials. This method has been tested on a system with a cooled CCD camera as the parallel detector and illustrated by the analysis of low concentration Ca in an organic matrix. The method is expected to be generally applicable to spectral analysis affected by gain variations of parallel detectors.

Calcium↗

PubMed1999

Calcium Quantitation with a Parallel Electron Energy Loss Spectroscopy/Cooled Charge-Coupled Device/200 keV System.

: We evaluated factors affected the accuracy and precision of quantitating trace concentration of Ca with electron energy loss spectroscopy (EELS). These factors include internal reflection in the spectrometer, precision of correlation between standards and experimental spectra, and radiation damage-induced spectral changes. We present methods of correcting for these effects and improving the reliability of trace Ca quantitation. A two-step fitting procedure is described that improves the retrieval of small Ca signals from the large background common to biological specimens. After optimizing the experimental conditions and data processing procedures, our current system can detect about 2.2 mmol/kg Ca in a 730-Å thick specimen at a total dose of about 410 nA. sec at 95% confidence level by fitting the first difference spectra. Because of the 0.1% residual gain variation after gain normalization, the first difference spectrum fitting is still the preferred method for trace Ca quantitation. Our study also demonstrates the clear advantage of using a 200 keV system, instead of 120 keV or lower accelerating voltages, for EELS analysis of relatively thick biological cryosections.

Journal Article↗

PubMed1999

Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes.

We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 microM), and production of ROS was measured using 2', 7'-dichlorofluorescin diacetate. Data were expressed as means +/- SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 +/- 2% (n = 6, P < 0.05) ACh 21 +/- 5% (n = 6, P < 0.05) vs controls 42 +/- 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 +/- 0.16 (n = 7, P < 0.05) vs. controls, 0.16 +/- 0. 03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 +/- 7% (n = 5), 2-MPG+ACh 47 +/- 6% (n = 6); ROS signals: 5-HD+ACh 0.09 +/- 0.03 (n = 5), 2-MPG+ACh 0.01 +/- 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 +/- 0.07, n = 5). These results demonstrate that activation of mitochondrial K(ATP) channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.

Acetylcholine↗

PubMed1999

Role of GABAB receptor-mediated inhibition in reciprocal interareal pathways of rat visual cortex.

In neocortex, synaptic inhibition is mediated by gamma-aminobutyric acid-A (GABAA) and GABAB receptors. By using intracellular and patch-clamp recordings in slices of rat visual cortex we studied the balance of excitation and inhibition in different intracortical pathways. The study was focused on the strength of fast GABAA- and slow GABAB-mediated inhibition in interareal forward and feedback connections between area 17 and the secondary, latero-medial visual area (LM). Our results demonstrate that in most layer 2/3 neurons forward inputs elicited excitatory postsynaptic potentials (EPSPs) that were followed by fast GABAA- and slow GABAB-mediated hyperpolarizing inhibitory postsynaptic potentials (IPSPs). These responses resembled those elicited by horizontal connections within area 17 and those evoked by stimulation of the layer 6/white matter border. In contrast, in the feedback pathway hyperpolarizing fast and slow IPSPs were rare. However weak fast and slow IPSPs were unmasked by bath application of GABAB receptor antagonists. Because in the feedback pathway disynaptic fast and slow IPSPs were rare, polysynaptic EPSPs were more frequent than in forward, horizontal, and interlaminar circuits and were activated over a broader stimulus range. In addition, in the feedback pathway large-amplitude polysynaptic EPSPs were longer lasting and showed a late component whose onset coincided with that of slow IPSPs. In the forward pathway these late EPSPs were only seen with stimulus intensities that were below the activation threshold of slow IPSPs. Unlike strong forward inputs, feedback stimuli of a wide range of intensities increased the rate of ongoing neuronal firing. Thus, when forward and feedback inputs are simultaneously active, feedback inputs may provide late polysynaptic excitation that can offset slow IPSPs evoked by forward inputs and in turn may promote recurrent excitation through local intracolumnar circuits. This may provide a mechanism by which feedback inputs from higher cortical areas can amplify afferent signals in lower areas.

Animals↗

PubMed1999

AFM tips: how sharp are they?

From both simple estimates and a 'blind' reconstruction based on cryo-AFM images of filamentous actin, we find that the radius of curvature at the apex of Si3N4 tips can be as small as 1 nm with a conical angle in the range 30 approximately 40 degrees, revealing a relatively high aspect ratio that is much greater than previously anticipated. Our results show that commercially available cantilevers are often sharp enough for routine high resolution imaging of biological materials, and suggest that factors other than an inherent blunt tip are probably responsible for frequent occurrences of poor resolution.

Actins↗

PubMed1998

Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes.

Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 +/- 3% to 14 +/- 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased approximately 2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 micromol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.

Animals↗