Biomedical subjects

Z Cao

Publications and source records attributed to Z Cao.

At least 145 records · Page 8Linked to original sources

PubMed1997

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein.

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-kappaB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-kappaB activation.

Animals↗

PubMed1997

IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and IkappaB kinase-alpha (IKK-alpha). A widely expressed protein kinase was identified that is 52 percent identical to IKK-alpha. IkappaB kinase-beta (IKK-beta) activated NF-kappaB when overexpressed and phosphorylated serine residues 32 and 36 of IkappaB-alpha and serines 19 and 23 of IkappaB-beta. The activity of IKK-beta was stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-alpha and IKK-beta formed heterodimers that interacted with NIK. Overexpression of a catalytically inactive form of IKK-beta blocked cytokine-induced NF-kappaB activation. Thus, an active IkappaB kinase complex may require three distinct protein kinases.

Amino Acid Sequence↗

PubMed1997

Biphasic binding kinetics between FepA and its ligands.

The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.

Bacterial Outer Membrane Proteins↗

PubMed1997

Identification and characterization of an IkappaB kinase.

Activation of the transcription factor NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-kappaB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-kappaB activation. CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells. CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway. This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation.

Cloning, Molecular↗

PubMed1997

Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

Adult↗

PubMed1997

Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria.

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.

Bacterial Outer Membrane Proteins↗

PubMed1997

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.

Amino Acid Sequence↗

PubMed1997

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.

Animals↗

PubMed1997

Nucleation and polymerization of sickle hemoglobin with Leu beta 88 substituted by Ala.

We have measured the solubility, and the rates of homogeneous and heterogeneous nucleation on sickle hemoglobin (HbS beta 6 Glu-->Val) additionally modified by site-directed mutagenesis to possess Ala rather than Leu at beta 88, which forms part of the receptor site for beta 6 Val in the sickle polymer. The solubility of the hemoglobin is increased at all temperatures, and is about 29 g/dl at 25 degrees C. Polymerization kinetics, induced by laser photolysis and observed by light-scattering intensity, showed exponential growth with rates about 300 times slower than experiments done on similar concentrations of HbS. When polymerization is carried out in small volumes, the time of measurable light-scattering signal to reach one-tenth of its final value (denoted as the tenth time) showed stochastic fluctuations, as is seen in pure HbS. Homogeneous nucleation rates were measured by observing distributions of tenth times and these rates were slowed by the mutation by almost 1000-fold relative to pure HbS. The kinetics, including the exponential progress curves and shape of the tenth time distributions, are well described by the double nucleation mechanism for polymerization. Analysis of the homogeneous nucleation rates leads to the surprising conclusion that the mutation has scarcely changed the energy of the intermolecular contacts despite the increase in solubility of the double mutant. This conclusion is supported by the stereochemistry of the modified contact site, in which the amount of exposed hydrophobic surface appears to be unchanged by the mutation. The increased solubility must therefore result from decreased motional freedom of molecules within the polymer, which could arise from tighter packing into the enlarged receptor pocket. This points up the ability of kinetic analysis to reveal important thermodynamic properties of assembly, and underlines the importance of the vibrational degrees of freedom in setting the final equilibrium constant. Chemical modifications to restrict vibrations and enhance the cost of polymerization may prove useful in constructing compounds to act as inhibitors of sickle cell gelation.

Binding Sites↗

PubMed1997

Technical considerations for optimal orthopedic imaging.

Over the past 25 years bone scintigraphy has played an essential role in most Nuclear Medicine departments, accounting for 25% to 60% of the patient volumes. No longer is every bone scan ordered as part of a metastatic workup. Today radionuclide bone imaging (RNBI) is included in most orthopedic diagnostic pathways. It is necessary to optimize the bone scan procedure to be sensitive, to carefully localize abnormal uptake for diagnosis, to correlate the bone scans, plain radiographs, computed tomographic (CT) scans, and magnetic resonance (MR) scans that accompany the patient, and to be precise in reporting a final impression. This can be done through meticulous attention to the image acquisition so that it encompasses not only equipment specifications and acquisition parameters, but also patient history, preparation, imaging protocols, positioning, and image correlation. The importance of these considerations, the rationale to explain them, and suggested guidelines for their implementation will be discussed in this article.

Bone Diseases↗

PubMed1997

Homogeneous nucleation in sickle hemoglobin: stochastic measurements with a parallel method.

The homogeneous nucleation rate for sickle hemoglobin polymerization has been measured for concentrations from 3.9 to 4.9 mM and temperatures from 13 degrees C to 35 degrees C by observing the stochastic fluctuations of the time to complete 10% of the reaction after photolysis of the carboxy derivative. To allow efficient data collection, a mesh was used to divide the photolysis beam into an array of smaller beams, which allowed parallel observation of about 100 different regions. Nucleation rates measured here are consistent with more restricted previously published data and, when combined with directly measured monomer addition rates, are consistent with previous analysis of progress curves. By describing these rates with equilibrium nucleation theory, the concentration of nuclei and hence their stability can be ascertained. Consequently, the chemical potential by which a monomer is attached to the polymer is determined. This attachment energy ranges from -6.6 to -8.0 kcal/mol between 15 degrees C and 35 degrees C. The enthalpic part of that chemical potential is found to be equal to the enthalpy determined by solubility measurements, as expected from thermodynamic considerations. The entropic portion of the contact chemical potential contributes from -21.4 to -8.7 kcal/mol. The vibrational chemical potential of monomers in the polymer ranges from -25.7 to -27.4 kcal/mol over the same temperatures.

Calorimetry↗

PubMed1997

Differential effects mediated by GABAA receptors in thalamic nuclei in lh/lh model of absence seizures.

Absence seizures represent synchronized burst-firing of thalamocortical neurons, which are driven by tonic GABAergic output of nucleus reticularis thalami (NRT). Activation of GABAA receptors on NRT neurons reduces NRT output and retards thalamocortical burst-firing. Although this mechanism in NRT may underlie antiabsence effects of benzodiazepines, it does not explain observations that barbiturates can worsen absence-seizures. In this study we tested the hypothesis that clonazepam and phenobarbital produce differential effects on GABAA receptors in the lh/lh genetic model of absence seizures after microinjection into NRT compared to VLa, a prototypic relay nucleus containing thalamocortical neurons. In NRT, phenobarbital (16-1600 nmol/cannula), clonazepam (160-2200 pmol/cannula) and muscimol (8.8-263 pmol/cannula) significantly suppressed absence seizure frequency. In VLa, phenobarbital (1.6 nmol) and muscimol (0.88 pmol) increased seizure frequency, whereas higher doses (160 nmol and 88 pmol, respectively) significantly suppressed seizure frequency. In contrast, clonazepam produced no effect on seizure frequency even at a dose of 2.2 nmol; this same dose significantly suppressed absence seizures after microinjection into NRT. These findings suggest that activation of GABAA receptors in NRT may suppress absence seizures, and that phenobarbital may worsen absence seizures through actions on GABAA receptors in thalamocortical cells (VLa). Region-specific GABAA receptor isoforms may underlie the contrasting effects of clonazepam after microinjection into NRT and VLa.

Animals↗

PubMed1997

MyD88: an adapter that recruits IRAK to the IL-1 receptor complex.

IL-1 is a proinflammatory cytokine that signals through a receptor complex of two different transmembrane chains to generate multiple cellular responses, including activation of the transcription factor NF-kappaB. Here we show that MyD88, a previously described protein of unknown function, is recruited to the IL-1 receptor complex following IL-1 stimulation. MyD88 binds to both IRAK (IL-1 receptor-associated kinase) and the heterocomplex (the signaling complex) of the two receptor chains and thereby mediates the association of IRAK with the receptor. Ectopic expression of MyD88 or its death domain-containing N-terminus activates NF-kappaB. The C-terminus of MyD88 interacts with the IL-1 receptor and blocks NF-kappaB activation induced by IL-1, but not by TNF. Thus, MyD88 plays the same role in IL-1 signaling as TRADD and Tube do in TNF and Toll pathways, respectively: it couples a serine/threonine protein kinase to the receptor complex.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Compensation for displacement of the focal point in cone beam single photon emission computed tomography reconstruction.

This study examined the effects of focal point displacement on image quality in cone beam single photon emission computed tomography (SPECT). A new image reconstruction algorithm that accounts for the focal point shift was derived and three shift geometries were investigated. The geometries included a lateral shift with a fixed focal length but off-center focusing, a linear axial shift with a variable focal length that depends linearly on the distance between a bin of the detector and the center of the detector, and a random axial shift with a randomly varying focal length. Computer simulation was conducted to evaluate the shift effects with a phantom that was composed of 118 small spherical sources. The results demonstrated that the lateral shift of the focal point was more critical to image quality than was the axial shift. With a 0.64 cm (1 pixel) lateral shift, noticeable artifacts was observed, while an axial shift resulted in minimal changes in image quality until it reached 8 cm (12.5 pixels). The derived reconstruction algorithm eliminated most of the artifacts caused by a fixed lateral shift or a linear axial shift of the focal point, but failed to do so for a random axial shift since the linear distribution assumed in image reconstruction did not match the random shift occurred in acquisition of the data.

Algorithms↗

PubMed1997

A triple-head SPECT system with parallel-hole collimators of different acceptance angles.

We proposed to use three different parallel-hole collimators for a triple-head SPECT system. One of the collimators had a small collimator acceptance angle to provide ultra-high spatial resolution and the other two had larger collimator acceptance angles to achieve high counts. A new 2D reconstruction algorithm that combined the data acquired from different collimators was derived to take advantages of both high resolution and high sensitivity. The algorithm was evaluated using a computer-simulated matrix of spherical sources. For noise-free data, the accuracy (mainly determined by spatial resolution) obtained from combination of the collimator acceptance angles of 1.35, 4.05 and 6.75 degrees (or 1.35, 5.40 and 9.45 degrees) was slightly inferior to that obtained from three same LEHR collimators (with a 2.70 degrees collimator acceptance angle). This is because the modulation transfer function (MTF) resulting from three different collimators decreases more quickly at low frequencies but becomes comparable at high frequencies as compared with the MTF of the 2.70 degrees collimator. With noisy data, however, the image quality obtained with three different collimators was better than that resulting from any combinations of three same collimators. The improvement was only achieved by using the derived algorithm, while the conventional FBP algorithm did not improve image quality even with the same collimator configuration.

Algorithms↗

PubMed1997

Renal amylin binding in normotensive and hypertensive rats: effects of angiotensin converting enzyme inhibition with perindopril.

OBJECTIVES: To investigate the effect of angiotensin converting enzyme inhibition with perindopril on the binding density of [125I]-rat amylin in the renal cortex in normotensive Sprague-Dawley rats, renally ablated hypertensive rats and spontaneously hypertensive rats. DESIGN: Sprague-Dawley rats, renally ablated hypertensive rats and spontaneously hypertensive rats were administered either the angiotensin converting enzyme inhibitor perindopril or no treatment. METHODS: The density of [125I]-rat amylin binding was measured in the renal cortex using autoradiography in vitro. The systolic blood pressure was measured by indirect tail-cuff plethysmography. The plasma renin activity was measured by radioimmunoassay. RESULTS: The density of [125I]-amylin binding was reduced by approximately 50% in Sprague-Dawley and subtotally nephrectomized Sprague-Dawley rats after treatment with perindopril. These changes were associated with a reduction in systolic blood pressure and an increase in plasma renin activity. In contrast, amylin binding in the perindopril-treated spontaneously hypertensive rats was not reduced, despite the prevention of a rise in systolic blood pressure and an increase in plasma renin activity. CONCLUSIONS: These findings provide further evidence for the hypothesis that there is an association among renal amylin binding, the renin-angiotensin system and blood pressure for rats of the Sprague-Dawley strain. In contrast, the lack of an effect of angiotensin converting enzyme inhibition on renal amylin binding for rats of the spontaneously hypertensive rat strain is consistent with previous findings that the changes in amylin binding in rats of this strain are not linked directly to the prevailing systemic blood pressure but may be associated with a developmental abnormality in the kidney of these rats.

Amyloid↗

PubMed1997

Pathogenesis of Acanthamoeba keratitis: carbohydrate-mediated host-parasite interactions.

Acanthamoeba keratitis is a sight-threatening corneal infection. In a recent study, the saccharide mannose has been shown to inhibit the binding of Acanthamoeba organisms to the epithelium of the cornea (L. D. Morton, G. L. McLaughlin, and H. E. Whiteley, Infect. Immun. 59:3819-3822, 1991). In an attempt to determine the molecular mechanism by which acanthamoebae adhere to the surface of the cornea, the present study was designed to determine whether Acanthamoeba castellanii derived from an infected human cornea (i) binds to mannose-containing glycoproteins (mannose-GPs) of corneal epithelium and (ii) expresses one or more mannose-binding proteins. Mannose-GPs of primary cell cultures of rabbit corneal epithelium were isolated by using three different agarose-conjugated, mannose-specific lectins. By electrophoresis blot-overlay assays, 35S-labeled acanthamoebae were shown to bind to mannose-GPs of corneal epithelium and to a neoglycoprotein, mannose-bovine serum albumin (mannose-BSA). 35S-labeled acanthamoebae also bound to microtiter wells coated with mannose-BSA in a concentration-dependent manner. The binding of amoebae to mannose-GPs was blocked by free methyl-alpha-D-mannopyranoside. The parasites did not bind to galactose-BSA or to many other proteins lacking mannose residues. A membrane-associated mannose-binding protein (136 kDa) of A. castellanii was isolated by affinity chromatography of detergent extracts of unlabeled parasites and of cell surface biotin-labeled parasites on a p-aminophenyl alpha-D-mannopyranoside-agarose column. The affinity-purified protein of the amoeba was shown to bind specifically to mannose-BSA. In summary, a mannose-binding protein is present on the surface membranes of Acanthamoeba, and corneal epithelial cells express Acanthamoeba-reactive GPs. One of the mechanisms of Acanthamoeba adhesion to the corneal surface may involve interactions between the mannose-binding protein of Acanthamoeba and mannose-GPs on the surface of corneal epithelium.

Acanthamoeba↗

PubMed1997

Increased density of renal amylin binding sites in experimental hypertension.

High-affinity binding sites for the pancreatic beta-cell hormone amylin have been reported in the kidney, and it has been postulated that these sites may be involved in the genesis of hypertension. In the present study, we have used in vivo injection of 125I-amylin and in vitro autoradiographic techniques to assess renal amylin binding in both a genetic and a surgically induced model of hypertension. In the spontaneously hypertensive rat (SHR) at 6 weeks of age, before the rise in systolic blood pressure, there was a 36% increase in density of amylin binding compared with their normotensive counterpart, the Wistar-Kyoto rat (WKY). In SHR, there was a further increase in the density of amylin binding (to 53% greater) as the systolic blood pressure rose between 6 and 12 weeks of age. Histological examination of kidneys from SHR at 12 weeks of age revealed staining for a brush border glycoprotein, normally restricted to the proximal tubules, extending from the urinary pole into half of the epithelial lining of the glomerular capsule. In contrast to WKY, these cells also bound 125I-amylin with high density in SHR. In a rat model of renal ablation and hypertension, systolic blood pressure correlated with the density of 125I-amylin binding in the renal cortex (r=.54, P=.003, n=28). The changes in amylin binding reported here suggest a possible role for this peptide and/or activation of its receptor in the genesis as well as the maintenance of hypertension.

Amyloid↗