Xu, wang, and Hu reply:
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Journal Article↗
Biomedical subjects
N Xu
Publications and source records attributed to N Xu.
NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP.
The inhibitor of apoptosis proteins (IAPs) regulate the caspase family of cysteine proteases, which play an important role in the execution of programmed cell death. Human X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases-3, -7, and -9. Here we show that the Bir3 domain is the minimal region of XIAP that is needed for potent caspase-9 inhibition. The three-dimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five alpha-helices, a three-stranded beta-sheet, and a zinc atom chelated to three cysteines and one histidine. The structure of the Bir3 domain is similar to that of the Bir2 domain of XIAP but differs from the previously determined structure of the Bir3 domain of MIHB. Based on site-directed mutagenesis, we have identified the regions of the Bir3 domain of XIAP that are important for inhibiting caspase-9. Despite the structural similarities of the Bir2 and Bir3 domain of XIAP, a different set of residues were found to be critical for inhibiting the individual caspases. These results suggest that XIAP inhibits caspase-3 and caspase-9 in a different manner.
Amino Acid Sequence↗
A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex.
mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.
Amino Acid Sequence↗
beta(2)-Integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice.
Interaction of CD11/CD18 beta(2) integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier. Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (Ancylostoma caninum), binds to the I domain of CD11a and CD11b and inhibits beta(2) integrin-dependent PMN adhesion. We describe a novel strategy using the endothelial cell-specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells. A construct containing NIF cDNA driven by the inducible endothelial cell-specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs). Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs. NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression. To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct. Analysis of lung tissue showed that intraperitoneal challenge of Escherichia coli resulted in NIF expression. Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by E coli challenge. These studies suggest the feasibility of conditionally blocking beta(2) integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.
Animals↗
Novel rapidity dependence of directed flow in high-energy heavy-Ion collisions
For high-energy nucleus-nucleus collisions, we show that a combination of space-momentum correlations characteristic of radial expansion, together with the correlation between the position of a nucleon in the nucleus and its stopping, results in a very specific rapidity dependence of directed flow: a reversal of sign in the midrapidity region. We support our argument by RQMD model calculations for Au+Au collisions at sqrt[s] = 200A GeV.
Journal Article↗
Reversibility of increased microvessel permeability in response to VE-cadherin disassembly.
We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.
Adherens Junctions↗
Temporal arteritis and fever: report of a case and a clinical reanalysis of 360 cases.
The purposes of this article are to report a case with temporal arteritis (TA) and to summarize and reanalyze the cases of temporal arteritis associated with fever in published articles for understanding better the clinical features of TA. A case with biopsy-proven TA is reported. The publications with TA and fever were searched by using MEDLINE in English from 1966 to 1999. Three hundred sixty cases of temporal arteritis associated with fever were reanalyzed. The results showed that a case of biopsy-proven TA with typically clinical manifestation was initially misdiagnosed and that the reanalysis of 360 cases revealed that the common clinical findings at presentation were abnormal temporal arteries, headache, low fever, loss of weight, polymyalgia rheumatica, jaw claudication, vision disorder, arthralgis or myalyias, and ear pain and that the uncommon clinical findings at presentation were high fever, malaise, anorexia, breast pain, transient ischemic attack/stroke, cough, mental disorder, diarrhea, and uterine prolapse, etc. Laboratory findings were the range of erythrocyte sedimentation rate (ESR) 14 to 149 with a mean of 97.0 mm/hr, white blood cells being normal or increased in the range of 10.9 to 22.9 x 10(9)/L, hemoglobin level 7 to 16 g/dL, the platelets count increased to 785 x 10(9)/L, and microscopic hematuria. The diagnosis was made by a combination of clinical features, an increased ESR, a response to steroids, and, most specifically, temporal artery biopsy. The initial diagnosis was misdiagnosed in 38.2% of patients. In conclusion, the features of TA associated with fever have not been widely appreciated yet. TA is a common cause of fever of unknown origin (FUO) in the elderly. TA should be considered when patients complain of common and uncommon manifestations. An elevated ESR will aid in the diagnosis of TA, and temporal artery biopsy will provide certainty.
Adult↗
A novel human apolipoprotein (apoM).
A novel human apolipoprotein designated apolipoprotein M (apoM) is described. The unique N-terminal amino acid sequence of apoM was found in an approximately 26-kDa protein present in a protein extract of triglyceride-rich lipoproteins (TGRLP). The isolated apoM cDNA (734 base pairs) encoded a 188-amino acid residue-long protein, distantly related to the lipocalin family. The mRNA of apoM was detected in the liver and kidney. Western blotting demonstrated apoM to be present in high density lipoprotein (HDL) and to a lesser extent in TGRLP and low density lipoproteins (LDL). The first 20 amino acid residues of apoM constituted a hydrophobic segment with characteristic features of a signal peptide. This was retained in the mature protein because of the lack of a signal peptidase cleavage site. In vitro translation in the presence of microsomes demonstrated translocation of apoM over the membrane and glycosylation but no signal peptide cleavage. The in vitro translated product remained associated with the microsomes after treatment with carbonate at pH 11, demonstrating that apoM is an integral protein. This finding suggests that apoM is linked to the single phospholipid layer of lipoproteins with a hydrophobic signal anchor. In conclusion, a novel human apolipoprotein, the function of which remains to be determined, is described.
Adult↗
NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP.
The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms. IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain. Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA). The structure of the BIR domain consists of a three-stranded antiparallel beta-sheet and four alpha-helices and resembles a classical zinc finger. Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3. The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs. Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme.
Amino Acid Sequence↗
MALDI-TOFMS of chemically modified recombinant hepatitis B surface antigen.
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Biotin↗
Electrospray ionization-mass spectrometry study of the interaction of cisplatin-adducted oligonucleotides with human XPA minimal binding domain protein.
Nucleotide excision repair (NER) is the process responsible for eliminating most ultraviolet (UV) radiation damage from DNA, as well as base alterations caused by a variety of mutagens. The xeroderma pigmentosum group A complementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that electrospray ionization-mass spectrometry (ESI-MS) can be an effective tool for the study of protein-DNA complexes. We have used ESI-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-adducted oligonucleotide and its interaction with the human XPA minimal binding domain (XPA-MBD). High-resolution FTICR experiments of the binding products showed that both double-stranded damaged 20-mer and double-stranded undamaged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding stoichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly preferential binding of XPA-MBD with the double-stranded damaged 20-mer compared to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spectrometry provides a fast and efficient approach for characterizing weak protein-DNA interactions such as the binding between XPA-MBD and a 20-mer oligonucleotide system.
Base Sequence↗
Inhibition of cardiovascular activity following microinjection of novel opioid-like neuropeptide nociceptin (orphanin FQ) into the rat rostral ventrolateral medulla.
Nociceptin (orphanin FQ), the newly discovered endogenous ligand for the novel opioid receptor-like 1 receptor, has been initially found to participate in pain modulation. In this study, centrally mediated cardiovascular actions of this peptide were investigated in the alpha-chloralose/urethane-anesthetized rats. We found that bilateral injection of nociceptin (10 nmol) into the rostral ventrolateral medulla (RVLM), wherein injection of excitatory amino acid dl-homocysteic acid (3 nmol) induced typical pressor responses, significantly reduced arterial blood pressure and heart rate by -32% and -15%, respectively. Reduction of blood pressure and heart rate in response to intra-RVLM injection of nociceptin was dose-dependent with a threshold dose being 3 nmol. Pretreatment with the selective nociceptin receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 (10 nmol), into the RVLM abolished the nociceptin-induced cardiovascular inhibition. In contrast, non-selective opioid receptor antagonist, naloxone (10 nmol), did not modify the hypotension and bradycardia induced by nociceptin, even though naloxone at the same dose prevented reduction of blood pressure and heart rate induced by intra-RVLM injection of methionine-enkephalin (3 nmol). Both [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 and naloxone injection alone had no significant effect on baseline blood pressure and heart rate. These data suggest that the newly discovered opioid-like neuropeptide nociceptin in the CNS exert powerful influence on hemodynamic activity by affecting the RVLM neurons. This influence is inhibitory in nature, which may not be active in normal physiological conditions. Moreover, the cardiovascular effects of nociceptin were mediated by activation of specific nociceptin receptors rather than typical naloxone-sensitive opioid receptors.
Animals↗
Persistent delivery of factor IX in mice: gene therapy for hemophilia using implantable microcapsules.
Severe hemophilia B is a life-threatening, life long condition caused by absence of or defective coagulation factor IX. Gene therapy could provide an alternative treatment to repeated injection of plasma-derived concentrate or recombinant factor IX. We have previously described the use of implantable microcapsules containing recombinant myoblasts to deliver human factor IX in mice. This study reports the generation of improved myoblast-specific expression vectors. Mouse myoblast clones transfected with the various vectors secreted factor IX in vitro, at rates between 70 and 1000 ng/10(6) cells/day. The recombinant myoblast clones were then encapsulated and implanted into mice. Immunocompetent mice implanted with encapsulated myoblasts had up to 65 ng of factor IX per milliliter in their plasma for up to 14 days, after which antibodies to human factor IX became detectable, and this coincided with decreased factor IX in mouse plasma. In immunodeficient mice, however, factor IX delivery was maintained at a constant level for at least 6 weeks (end of experiment). Interestingly, the highest-secreting myoblast clone in vitro did not deliver the highest level of hFIX in vivo. This discrepancy observed between performance in vitro and in vivo may have important implications for the development of gene therapy protocols based on recombinant cells.
Animals↗
The nociceptin receptor-mediated inhibition of the rat rostral ventrolateral medulla neurons in vitro.
The recently available antagonist selective for novel nociceptin receptor, [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2, was utilized in this study to verify specificity of nociceptin receptor in mediating the nociceptin-induced inhibition of electrical activity of neurons in the rostral ventrolateral medulla of rat brain slices. Perfusion of nociceptin (10 nM) considerably reduced spontaneously firing frequency of the medullary neurons. Co-perfusion of [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2 (10 microM) completely blocked the nociceptin-induced depression of the neuronal activity. Blocking effect of [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2 was concentration-dependent. However, the nociceptin antagonist did not modify basal, and opioid peptide enkephalin-depressed, firing rates of the neurons. In contrast to [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2, the non-selective opioid receptor antagonist naloxone (10 microM) failed to affect the nociceptin inhibition even though naloxone at a lower concentration (1 microM) readily blocked enkephalin-induced depression of the neuronal activity. These data indicate that the nociceptin-induced inhibition of spontaneous discharge of the rostral ventrolateral medulla neurons is specifically mediated by [Phe1 psi(CH2-NH)Gly2]NC(1-13)NH2-sensitive nociceptin receptors distinct from typical naloxone-sensitive opioid receptors.
Animals↗
Accurate measurement of H(N)-H(alpha) residual dipolar couplings in proteins.
A method for accurately measuring H(N)-H(alpha) residual dipolar couplings is described. Using this technique, both the sign and magnitude of the coupling can be determined easily. Residual dipolar coupling between H(N)(i)-H(alpha)(i) and H(N)(i)-H(alpha)(i-1) were measured for the FK506 binding protein complexed to FK506. The experimental values were in excellent agreement with predictions based on an X-ray crystal structure of the protein/ligand complex, suggesting that these residual dipolar couplings will provide accurate structural constraints for the refinement of protein structures determined by NMR.
Magnetic Resonance Spectroscopy↗
Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells.
Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by inference, changes in mRNA abundance are thought to affect only the stability of the mRNA, an assumption that is not always correct. Alternatively, direct measurements of mRNA decay are performed in a number of ways, including kinetic labeling techniques and administration of transcriptional inhibitors. Due to the nature of these techniques, they either are technically demanding or introduce a significant change in cell physiology. In addition, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. Here, we describe and discuss in detail two different transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated promoter systems as an effort to better elucidate the mechanistic steps and regulation underlying differential and selective mRNA turnover in mammalian cells. Both systems allow unequivocal monitoring of deadenylation and decay kinetics as well as determination of precursor-product relationship. In addition, decay rate constants and half-lives are determined and used in both methods to quantitatively denote the mRNA stability. Thus, they provide a reliable way to determine subtle yet physiologically meaningful changes in mRNA stability. Application of one method or the other covers the study of mRNA turnover in most mammalian cell types under a wide range of physiological conditions.
3T3 Cells↗
Characteristics of copper corrosion in simulated uterine fluid in the presence of protein.
In order to acquire more knowledge of the performance of copper-bearing intrauterine devices (Cu-IUDs), corrosion behavior of copper in a simulated uterine fluid was investigated in the presence of proteins. The proteins studied included serum albumin, gamma-globulin and hemoglobin. Electrochemical polarization resistance measurements indicated that, in all cases under study, the corrosion rate of copper declined with time and the proteins always increased the rate. Moreover, different kinds of protein showed different dependence of copper corrosion rate on protein level. Addition of serum albumin initially raised the copper corrosion rate; however, the higher albumin concentration resulted in less promotion of the corrosion. X-ray diffraction (XRD) of the specimen surface showed that, both in the presence and absence of serum albumin, cuprous oxide was the only corrosion product, even though at higher concentrations of albumin less cuprous oxide was formed. This implies that serum albumin does not alter the corrosion mechanism. Electrochemical cathodic reduction of the oxide film and chemical analysis of solution after corrosion testing proved that the proportion of soluble ionic copper in the whole corrosion products varied with the albumin level. The copper corrosion rate in the presence of gamma-globulin or hemoglobin increased monotonically with increasing concentration of the protein. These three proteins shifted the corrosion potential of copper towards negative. It could be inferred that the proteins accelerated the anodic dissolution process of copper and hence enhanced its corrosion.
Body Fluids↗
Characterization of AT2 receptor expression in NIH 3T3 fibroblasts.
1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts. 2. Characterization with selective ligands, dithiothreitol, and GTP gamma S, indicated that only the AT2 subtype was expressed. 3. AT2 receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT2 receptors being expressed earlier than ACE. In contrast, high expression of AT2 receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras. 4. Our results imply a possible relation of AT2 receptors to cell growth and cell-cell contact.
3T3 Cells↗