Biomedical subjects

M Zhou

Publications and source records attributed to M Zhou.

At least 217 records · Page 12Linked to original sources

PubMed1997

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology.

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.

Binding Sites↗

PubMed1997

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement.

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.

Animals↗

PubMed1997

Tn5 transposase mutants that alter DNA binding specificity.

Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition. We report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OE-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA. In addition, two of the Tnp mutants (TP47 and EK54) appear to be change-of-specificity mutants, since they alter the recognition of OE versus IE relative to the wild-type Tnp. EK54 enhances OE recognition but decreases IE recognition. TP47 enhances both OE and IE recognition but with a much greater enhancement for IE than for OE. This change-of-specificity effect of TP47 is observed only when TP47 Tnp is synthesized in cis to the DNA that contains the ends. We propose that Lys54 makes a favorable interaction with an OE-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IE-specific nucleotide pair(s) than it does with the corresponding OE-specific nucleotide pair(s). A model to explain the preference of TP47 Tnp for the IE in cis but not in trans is proposed.

Amino Acid Sequence↗

PubMed1997

The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70.

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.

Apolipoprotein B-100↗

PubMed1997

Elongation factor Ts of Chlamydia trachomatis: structure of the gene and properties of the protein.

A putative structural gene cluster containing four open reading frames (ORFs) located downstream of the omp1 gene of Chlamydia trachomatis mouse pneumonitis (MoPn) was cloned and sequenced. A GenBank survey indicated that the identified cluster is similar to the rpsB-tsf-pyrH(smbA)-frr region of Escherichia coli. The second ORF was 846 bp encoding a 282-amino-acid polypeptide with a calculated M(r) 30,824. Alignment of this deduced protein sequence and E. coli elongation factor Ts (EF-Ts, product of tsf) demonstrated 34% identity and an additional 14% similarity. The putative chlamydial tsf gene was expressed in E. coli as a nonfusion protein and as a 6x His-tagged fusion protein. By SDS-PAGE analysis, the molecular weights of the nonfusion recombinant protein and a protein of chlamydial elementary bodies (EBs), which was recognized by monoclonal antibodies derived from the nonfusion recombinant protein, are 34 kDa. The purified recombinant 6x His-tagged fusion protein increased the rate of GDP exchange with both Chlamydia and E. coli elongation factor Tu (EF-Tu). These data show that the second gene of the identified cluster is tsf. Unlike EF-Ts from any other species, its activity was comparable to that of E. coli EF-Ts in exchange reaction with E. coli EF-Tu.

Amino Acid Sequence↗

PubMed1997

A two-site model for ApoB degradation in HepG2 cells.

Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were co-treated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLN-sensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway.

Apolipoproteins B↗

PubMed1997

Endothelial nitric oxide synthase is downregulated during hyperdynamic sepsis.

Although studies have shown that endothelium-derived nitric oxide (NO) release is depressed during endotoxic shock or polymicrobial sepsis, it remains unknown whether the decreased release of endothelium-derived NO during the hyperdynamic stage of sepsis is due to downregulation of endothelial NO synthase. To study this, adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 10 h after CLP (i.e., hyperdynamic sepsis) or sham operation, the aorta was removed and a monoclonal antibody against endothelial (constitutive) NO synthase (E-NOS) was used to determine the immunohistochemical presence and electron microscopic localization of E-NOS in rat aortic endothelial cells. Image analysis was used to quantify aortic E-NOS. In additional groups of animals, the aorta was isolated at 10 h after CLP and the vascular responses to an endothelium-dependent vasodilator, acetylcholine, and an endothelium-independent vasodilator, nitroglycerine, were determined. The results indicate that the number of E-NOS negative endothelial cells increased from 7% in shams to 22% in septic animals. E-NOS densely labeled endothelial cells were significantly reduced from 20% to 8% at 10 h after CLP. The E-NOS positive area in aortic endothelial cells was reduced from 26.1 +/- 1.0 microm2/standard frame in sham to 22.3 +/- 0.9 microm2/standard frame in septic animals. Moreover, acetylcholine-induced but not nitroglycerine-induced vascular relaxation was significantly depressed at 10 h after the onset of sepsis. These results, taken together, indicate that the decreased E-NOS in the vascular endothelial cell is at least in part responsible for endothelial cell dysfunction (i.e., the reduced endothelium-derived NO release) observed during the early, hyperdynamic stage of polymicrobial sepsis.

Animals↗

PubMed1997

Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia.

Bcl-2 and its homologue, Bcl-x1, encode membrane-associated proteins that protect neoplastic cells from DNA damage-induced apoptosis, whereas Bax is a Bcl-2 antagonist that promotes cell death. In the present study, we examined the expression and regulation of these genes at both the mRNA and protein level in 22 pediatric acute lymphoblastic leukemia (ALL) cell lines, as well as their sensitivity to apoptosis after exposure to ionizing radiation (IR). Eleven of 22 lines expressed wild-type (wt) p53, 4 expressed mutant p53, and 7 did not express p53 (p53-null). Nine of 22 (41%) lines expressed Bcl-2; of these, 8 were wt-p53+ and 1 expressed mutant p53. Bcl-2 was not expressed in any p53-null lines. In contrast, all 22 lines were positive for Bcl-x1 and Bax, although expression level varied. Treatment with IR (10 Gy) induced both downregulation of Bcl-2 and upregulation of Bax at 2 to 5 hours post-IR in 5 of 8 (63%) wt-p53+ lines, leading to apoptosis. Conversely, lines that failed to both downregulate Bcl-2 and upregulate Bax after IR were resistant to apoptosis. Although levels of Bcl-x1 expression varied among the 22 lines, high levels of Bcl-x1 were observed in 5 of 7 (71%) p53- lines. There were no obvious changes in the expression of Bcl-x1 in these lines after IR. However, among the p53-null lines, resistance to IR was observed only in those expressing high levels of Bcl-x1. These results suggest that expression of Bcl-2 but not Bcl-x1 is p53-dependent and that IR-induced downregulation of Bcl-2 and upregulation of Bax occur in most wt-p53+ lines and are associated with radiosensitivity. Furthermore, high-level expression of Bcl-x1 occurs predominantly in p53-null lines and is associated with resistance to IR-induced apoptosis in these lines, indicating differential expression and regulation of Bcl-2 and Bcl-x1 in pediatric ALL.

Apoptosis↗

PubMed1997

Kit receptor dimerization is driven by bivalent binding of stem cell factor.

Most growth factors and cytokines activate their receptors by inducing dimerization upon binding. We have studied binding of the dimeric cytokine stem cell factor (SCF) to the extracellular domain of its receptor Kit, which is a receptor tyrosine kinase similar to the receptors for platelet-derived growth factor and colony-stimulating factor-1. Calorimetric studies show that one SCF dimer binds simultaneously to two molecules of the Kit extracellular domain. Gel filtration and other methods show that this results in Kit dimerization. It has been proposed that SCF-induced Kit dimerization proceeds via a conformational change that exposes a key receptor dimerization site in the fourth of the five immunoglobulin (Ig)-like domains in Kit. We show that a form of Kit containing just the first three Ig domains (Kit-123) binds to SCF with precisely the same thermodynamic parameters as does Kit-12345. Analytical ultracentrifugation, light scattering, and gel filtration show that Kit-123 dimerizes upon SCF binding in a manner indistinguishable from that seen with Kit-12345. These data argue that the fourth Ig-like domain of Kit is not required for SCF-induced receptor dimerization and provide additional support for a model in which bivalent binding of the SCF dimer provides the driving force for Kit dimerization.

Chromatography, Gel↗

PubMed1997

Stage of susceptibility to carcinogenicity of prenatal dietary fat exposure tested by blastocyst transfer.

A previous experiment with exposure of female mice to a diet high in fat from 4 weeks of age through to the end of pregnancy demonstrated a substantial increase in the frequency of reproductive system tumors in their female offspring. Over this wide period of exposure, one or more specific periods of sensitivity to the carcinogenic effects of dietary fat should exist. To test whether a high fat diet affects maternal germ cells and/or zygote development through to blastocyst formation, two blastocyst transfer experiments were performed. Blastocysts from a dam on high fat were transferred to a pseudopregnant dam on low fat. The reverse type of transfer was performed for contrast. These experiments did not reveal any significant effect of dietary fat on maternal germ cells or zygote development.

Adenocarcinoma↗

PubMed1997

Two EGF molecules contribute additively to stabilization of the EGFR dimer.

Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand-induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small-angle X-ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the hGH and interferon-gamma receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors.

Animals↗

PubMed1997

Crystal structure of bovine low molecular weight phosphotyrosyl phosphatase complexed with the transition state analog vanadate.

The early transition metal oxoanions vanadate, molybdate, and tungstate are widely used inhibitors for phosphatase enzymes. These oxoanions could inhibit such enzymes by simply mimicking the tetrahedral geometry of phosphate ion. However, in some cases, the enzyme-inhibitor dissociation constants (Ki) for these oxoanions are much lower than that for phosphate. Such observations gave rise to the hypothesis that in some cases these transition metal oxoanions may inhibit phosphomonoesterases by forming complexes that resemble the trigonal bipyramidal geometry of the SN2(P) transition state. As a test of this, the crystal structures of a low molecular weight protein tyrosine phosphatase at pH 7.5 complexed with the inhibitors vanadate and molybdate were solved at 2.2 A resolution and compared to a newly refined 1.9 A structure of the enzyme. Geometric restraints on the oxoanions were relaxed during refinement in order to minimize model bias. Both inhibitors were bound at the active site, and the overall protein structures were left unchanged, although some small but significant side chain movements at the active site were observed. Vanadate ion formed a covalent linkage with the nucleophile Cys12 at the active site and exhibited a trigonal bipyramidal geometry. In contrast, simple tetrahedral geometry was observed for the weaker molybdate complex. These studies are consistent with the conclusion that vanadate inhibits tyrosine phosphatases by acting as a transition state analog. The structure of the vanadate complex may be expected to closely resemble the transition state for reactions catalyzed by protein tyrosine phosphatases.

Animals↗

PubMed1997

Upregulation of endogenous p53 and induction of in vivo apoptosis in B-lineage lymphomas of E(mu)-myc transgenic mice by deregulated c-myc transgene.

E(mu)-myc transgenic mice carry a constitutively overexpressed c-myc oncogene and develop B-lineage lymphomas. Previous studies have shown that c-myc overexpression can lead to in vitro apoptosis. Here, we investigated the in vivo effects of altered c-myc expression on cell proliferation versus death in spontaneously arising E(mu)-myc tumors. E(mu)-myc tumors display extensive in vivo apoptosis confined to small clusters of cells with greatly increased expression of both the c-myc transgene and the endogenous p53 gene as compared with that in normal, pretumor, or surrounding tumor tissue. This restricted overexpression of both the c-myc transgene and the endogenous p53 gene in small clusters of apoptotic tumor cells indicates that overexpression of these genes and apoptosis are not obligatory or uniform during tumor development and suggests that further somatic mutations or microenvironmental influences may be responsible for these properties. Nevertheless, the clear ability of tumor cells to undergo apoptosis in vivo may be exploitable for therapeutic purposes.

Animals↗

PubMed1997

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.

Arginine-tRNA Ligase↗

PubMed1997

Intracrine and autocrine effects of basic fibroblast growth factor in vascular smooth muscle cells.

In order to elucidate the effects of the different basic fibroblast growth factor (bFGF) isoforms on vascular smooth muscle, we examined aorta-derived vascular smooth muscle cells from transgenic mice expressing the human isoforms of bFGF. Four cell lines were examined from mice in which transgene expression was driven by the ubiquitous phosphoglycerate kinase promoter. Overexpression and cellular localization was confirmed by Western blot analysis in vascular smooth muscle cells from mice expressing: all four human bFGF isoforms (24, 22, 21, and 18 kDa); all three nuclear targeted isoforms (24, 22, and 21 kDa); only the 24 kDa isoform; and the only secreted/non-nuclear targeted isoform, 18 kDa. All lines showed approximate four-fold increases in bFGF expression, nuclear localization of all nuclear targeted bFGF isoforms, and cytosolic localization of only the 18 kDa bFGF. Measurement of [3H]thymidine incorporation into quiescent cells stimulated with increasing concentrations of serum, showed increased DNA synthesis in cell lines expressing any bFGF isoform when compared to non-transgenic control cells, and a further increase in DNA synthesis in cells expressing the nuclear targeted isoforms (24, 22, and 21 kDa) over the 18 kDa bFGF expressing cell line at any concentration of serum. All cells showed equal label incorporation when stimulated with 10 ng/ml of platelet-derived growth factor confirming an equal potential for DNA synthesis. Neutralizing the bFGF antibody markedly decreased serum-stimulated DNA synthesis, but only in the cell lines overexpressing the secreted/non-nuclear targeted 18 kDa isoform. These results suggest amplification of DNA synthesis through synergistic intracrine and autocrine effects of the nuclear targeted and non-nuclear targeted bFGF isoforms in vascular smooth muscle cells.

Animals↗

PubMed1997

A new MHC locus that influences class I peptide presentation.

We have investigated the HLA-B27-restricted CTL response to HY minor histocompatibility antigens in rats and mice transgenic for HLA-B27 and human beta2-microglobulin. A polymorphism was found at a locus within the H2 complex, producing two distinct but overlapping sets of B27-presented HY peptides. The locus, named Cim2, mapped between the K and Pb loci, and its product is therefore distinct from TAP, LMP, and tapasin. Identical findings in rats and mice, including identical HY peptide sequences and the failure of a rat Tap2A transgene to alter CTL recognition, suggest that a homologous locus with similar polymorphism exists in the rat. Cim2, or a closely linked locus, was found to exert a broad effect on peptide loading of both HLA-B27 and mouse class I alleles. The data thus establish a strong, previously unrecognized MHC-encoded influence on the class I antigen pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Expression of interleukin-6 receptors by pediatric acute lymphoblastic leukemia cells with the t(4;11) translocation: a possible target for therapy with recombinant IL6-Pseudomonas exotoxin.

We have detected expression of interleukin-6 receptors (IL-6R) by primary leukemic cells from three of six patients with t(4;11)+ ALL. Scatchard analysis revealed from 960 to 2100 high-affinity IL-6R/cell on these cells (median, 1560; mean, 1540). All three IL-6R+ cases also expressed CD33, which was not expressed on IL-6R-negative cases. To determine if these receptors could serve as a target for a recombinant ligand-toxin, we examined the sensitivity of primary IL-6R+ ALL cells to a recombinant IL6-Pseudomonas exotoxin (IL6-PE4E) fusion protein, in which the toxicity and specificity of the chimeric toxin was enhanced by substitution of four glutamine residues for naturally occurring amino acids in PE domain I. Primary cells from IL-6R+ cases were sensitive to IL6-PE4E in a 48-h cytotoxicity assay, with ID50 values (concentrations causing 50% decrease in viability) ranging from 23 ng/ml to 92 ng/ml (median, 61; mean, 58). Furthermore, incubation of these cells with 10(3) ng/ml IL6-toxin for 24 h prevented their subsequent engraftment in SCID mice. Thus, IL6-PE4E may be useful for ex vivo purging of IL-6R+ leukemic cells in an autologous bone marrow transplantation setting and possibly for therapy of residual, chemotherapy-resistant disease.

ADP Ribose Transferases↗

PubMed1997

Real-time multichannel computerized electrogastrograph.

The purpose of this study was to develop a real-time multichannel computerized electrogastrograph (EGG) to measure and analyze electrical signals from the human abdominal surface. A soft-contact matrix composed of 25 cutaneous electrodes is embedded evenly in a latex mat. The mat can be firmly attached to the abdominal surface by drawing a vacuum between the matrix and the stomach. Twenty-five high-amplification filter/amplifiers provide a high signal-to-noise ratio and flat amplitude response for a signal between 0.02 and 0.12 Hz (1.2-7.2 cpm). The computer program provides waveform and frequency analysis for any chosen channel and mapping analyses for all 25 channels. A two-dimensional propagation exploration program was also developed. Using four different mapping analysis program subroutines, the optimal points for analyzing the EGG signals can be reliably found and variability of these locations can be observed easily. Results show differences in the EGG mappings of normal and abnormal subjects.

Adult↗