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Biomedical subjects

Ying Yu

Publications and source records attributed to Ying Yu.

At least 73 records · Page 4Linked to original sources

The effects of alphaGalCer-induced TCRValpha24 Vbeta11(+) natural killer T cells on NK cell cytotoxicity in umbilical cord blood.

PURPOSE: The first objective of this study was to investigate in vitro effects of alpha-galactosylceramide (alphaGalCer) on the proliferation of umbilical cord blood (UCB) natural killer T (NKT) cells and enhancement of their cytotoxicity. The second one is to examine whether purified NKT cells could affect the cytotoxicity of UCB-NK cells either in the presence or absence of dendritic cells (DCs). METHODS: Mononuclear cells (MNCs) from UCB were cultured for 2 weeks in the presence of IL-2 (100 U/ml), with or without alphaGalCer. The effect of neutralizing monoclonal antibodies (MoAb) against TCRValpha24 and CD1d was also examined. TCRValpha24 Vbeta11 double positive NKT cells were purified by FACS sorter and then cocultured with syngeneic isolated UCB(-)CD56(+)NK cells in either the presence or absence of DCs. The cytotoxicity against various malignant cell targets and cytokine production was determined. RESULTS: The addition of alphaGalCer induced human NKT cells to proliferate in UCB-MNCs to a greater extent than in adult PB-MNCs. However, it suppressed the cytotoxic activity against malignant cell targets. Anti-TCRValpha24 and CD1d MoAb recovered the cytotoxicity by inhibiting the proliferation of UCB-NKT cells. NKT cells cocultured with auto-DCs significantly increased NK cell cytotoxicity against K562, and Raji cells and produced IFN-gamma at much higher levels than UCB-NKT cells alone. CONCLUSION: In UCB samples, alphaGalCer-pulsed DCs and NKT cells acted together to enhance NK cytotoxicity in vitro.

Adult↗

Differential expression of CD146 in tissues and endothelial cells derived from infantile haemangioma and normal human skin.

Haemangioma is the most common tumour of endothelial origin, occurring in 4-10% of Caucasian infants. It is characterized by rapid growth during the first year of postnatal life, followed by spontaneous regression from 1 to 7 years of age. The cell surface adhesion molecule CD146 has been identified as an endothelial cell marker. Despite advances in understanding the functional role of CD146 in normal endothelial cells and tumour progression, its expression and a possible role in an endothelial tumour have not been studied. As part of an investigation of endothelial cell alterations in infantile haemangioma, differential expression studies were performed with several known antigens and endothelial cell markers. Using immunohistochemical and flow cytometric analyses, cultured human dermal microvascular endothelial cells isolated from newborn foreskin (HDMEC) were compared with endothelial cells derived from haemangioma tissue (HemECs). In addition, immunohistochemistry was used to compare haemangioma tissues with normal human skin. Unexpectedly, cultured HemECs showed a significantly lower level of CD146 than HDMECs by both flow cytometric analysis and immunofluorescence staining. Using immunohistochemical studies, it was further demonstrated that endothelia in all haemangioma tissues, regardless of the tumour phase, showed negative immunoreactivity for CD146. In contrast, strong positive staining for CD146 was observed in the pericyte-like cells that surround the endothelial layers. These findings are believed to be relevant to the molecular basis of haemangioma. Furthermore, it is possible that antibodies against CD146 may be useful for separating haemangioma-derived endothelial cells from normal endothelial cells and pericytes.

Antigens, CD↗

HIV vector production mediated by Rev protein transduction.

HIV-1-based vectors are promising tools for gene therapy because of their ability to integrate into nondividing cells. Their safety in clinical applications remains a major concern. Recombination events occurring among plasmid constructs during vector production could potentially lead to the generation of replication-competent viruses. The safety of HIV-1-based vectors can be improved by removing all regions of the viral genome that are not absolutely required for vector production or function. In this study, we demonstrate that the HIV-1 rev gene is dispensable for the production of HIV-1-based vectors if the vector-producing cells are supplied with purified Rev protein. We compared the efficiency of vector production among Rev, TAT-Rev (Rev fused to the protein transduction domain of the HIV TAT protein), and Rev/Pep-1 (Rev complexed with the carrier peptide Pep-1). Our results showed that 293T cells efficiently internalized TAT-Rev and Rev/Pep-1 and high-titer vector preparations were obtained with this approach. Vectors generated by such an approach showed little difference in their efficiencies of transduction of established cell lines and primary cells compared with vectors generated by standard plasmid cotransfection. Eliminating the requirement for the HIV-1 rev gene during vector production should improve the safety of applying HIV vectors in human clinical trials.

Animals↗

Structure-activity of valencenoid derivatives and their repellence to the Formosan subterranean termite.

Eight valencenoid derivatives were evaluated for their repelling activity against Formosan subterranean termites, Coptotermes formosanus Shiraki. Among them, 1,10-dihydronootkatone was the strongest repellent, and valencene was the weakest. Results of the structure-repellency relationships indicated (1) reduction of the ketone group to the alcohol on position 2 of nootkatone curtailed the activity; (2) because of the low activity of valencene relative to nootkatone that the ketone group was essential for repellent activity; (3) reduction of the 1,10 double bond (1,10-dihydronootkatone and tetrahydronootkatone) produced compounds more repellent than nootkatone; (4) the isopropenyl group probably does not participate in binding as evidenced by no significant difference in the repellent activity among nootkatone (double bond between position 11 and 12), isonootkatone (double bond between position 7 and 11), and 11,12-dihydronootkatone.

Animals↗

PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression.

Capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) (SOC) channels plays an important role in returning Ca(2+) to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). A rise in [Ca(2+)](cyt) and sufficient Ca(2+) in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5-48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca(2+) from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca(2+)](cyt), facilitates return of Ca(2+) to the SR, and enhances PASMC growth.

Animals↗

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells.

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.

Apoptosis↗

[Expression of BRD7-interacting proteins,BRD2 and BRD3, in nasopharyngeal carcinoma tissues].

BACKGROUND & OBJECTIVE: BRD7 is a novel gene tightly associated with nasopharyngeal carcinoma(NPC) cloned by cDNA representational difference analysis (cDNA RDA). Two proteins,BRD2 and BRD3, including bromodomain and interacting with BRD7 protein had been screened from human fetal brain cDNA library by yeast two-hybrid system. This study was designed to further identify the interactions of BRD2 and BRD3 with BRD7 respectively and to investigate the expression and action pattern of BRD2 and BRD3 in NPC. METHODS: BRD2 and BRD3 genes were respectively co-transformed to yeast Y187 with BRD7 gene, then the yeast cotransformers were blotted to nylon membrane. And then the expression of report gene Lac Z by beta-Gal was determined and the interactions of BRD2 and BRD3 proteins with BRD7 protein were identified. Besides,reverse transcription-polymerase chain reaction (RT- PCR) was used to examine the expression of BRD2 and BRD3 genes in normal nasopharyngeal epithelium and NPC biopsies, and to detect the effect of re-expression of BRD7 gene on the expression of BRD2 and BRD3 genes in HNE1 stably transfected BRD7 gene. RESULTS: The yeast transformers showed all blue by yeast two-hybrid system, which further identified that BRD2 and BRD3 proteins could respectively interact with BRD7 protein. Down-expression or loss of BRD2 and BRD3 genes were detectable in NPC biopsies. The expression levels of BRD2 and BRD3 were increasing with the re-expression of BRD7 gene in HNE1 stably transfected with BRD7. CONCLUSION: BRD7 protein could respectively interact with proteins, BRD2 and BRD3, and BRD7 could up-regulate the expression levels of BRD2 and BRD3 genes in mRNA level to some extent. Each of these three homolog proteins might be capable of forming heteromers with the others, which play important roles in the suppression of growth of NPC cells.

Chromosomal Proteins, Non-Histone↗

[Clinical features and therapy of 106 cases of severe acute respiratory syndrome].

OBJECTIVE: To investigate the clinical characteristics of severe acute respiratory syndrome (SARS) and find out its effective treatment. METHODS: A total of 106 cases of SARS were analyzed prospectively. RESULTS: In this group, 56 were male and 50 female, aged from 15 to 81 years [average (36 +/- 10) years]. Common symptoms included fever (98.1%), chills (75.5%), cough (71.7%), headache and breathless (both 43.4%), diarrhea (24.5%) and rare rales in the lungs (11.2%). Laboratory test showed leukopenia (34.0%), lymphopenia (81.1%) and an extraordinary decrease of CD(4)(+) T cells (98.1%). Other rare abnormalities included liver injury (elevated alanine aminotransferase in 7.6%) and thrombocytopenia (3.8%). Almost all patients suffered from hypoxemia (PaO(2) less than 90 mm Hg in 90.2%, less than 70 mm Hg in 28.6%). Chest radiographs showed that unilateral focal patchy involvement in 34.0% of the patients, and unilateral multifocal or bilateral involvement were 11.3% and 46.2% respectively. Treatment regimens included small doses of steroids (methylprednisolone 40-80 mg, q12 h recommended) accompanied with broad-spectrum antibiotics such as the second generation of cephalosporins and macrolides and some other antiviral drugs. Meanwhile, emphasis was placed on oxygen support and coping with their underlying diseases. CONCLUSIONS: SARS has various presentations of clinical features and laboratory tests. Detection of CD(4)(+) T cell count is beneficial to diagnose SARS in early stage. Effective treatment includes various regimens, oxygen support and small doses of steroids.

Adolescent↗

[Effects of selenium on soybean chloroplast ultra-structure and microelement content of soybean leaves under continuous cropping stress].

Pot and field experiments were carried out to study the effect of low Se concentration on soybean chloroplast ultra-structure and on Mg, Fe, Mn and contents in soybean leaves under continuous cropping stress. The results showed that Se could certainly increase the contents of Mg, Fe and Mn in soybean leaves when the application rate of Se was below 0.50 mg.kg-1 in pot experiment, and below 300 g.hm-2 in field experiment. In pot experiment, Se concentration positively correlated with Mg and Fe contents in soybean leaves, but the relationship between Mn and Se was hardly to be quantified. Transmission electron microscopic observation indicated that under continuous cropping stress, application of Se could hold the soybean chloroplast membrane structure in good condition. When Se was not applied, the chloroplast swelled, and its grana disappeared, and even, transformed into etioplast. These experiments indicated that application of low Se could alleviate peroxidative stress resulting from continuous cropping, and keep the soybean grow normally.

Chloroplasts↗

[Cu adsorption by phaeozem and burazem].

Studies on the thermodynamics and kinetics of copper (Cu2+) adsorption by phaeozem and burozem showed that that the adsorption of Cu2+ by these two soils was increased with the increasing concentration of Cu2+ within the range of Cu2+ concentrations used in the experiment, but the amount of Cu2+ adsorbed and fixed on phaeozem was much higher than that on burozem. When the test Cu2+ concentration in adsorption equilibrium was 95 mg.kg-1, its adsorption by burozem was nearly 3720 mg.kg-1, whereas by phaeozem was as high as 6076 mg.kg-1. Under the highest CuCl2 treatment of 400 mg.kg-1, Cu2+ adsorbed by phaeozem and burozem was 6159.0 mg.kg-1 and 4736.6 mg.kg-1, respectively. The adsorption of Cu2+ by the test soils could significantly conform to the Freundlich and Temkin equation, while Langmuir equation was not applicable to describe the isothermal adsorption processes of Cu2+ by the two soils. The adsorption of Cu2+ by the test soils was very rapid, which could reach its balance after 15-20 min. The adsorption within the first 2 min at its beginning stage accounted for 90% of the Cu equilibrium adsorption. The adsorptive rate of Cu2+ by phaeozem was decreased more quickly than that by burozem with the time. The two-constant equation was the optimal model to describe the adsorption of Cu2+. The other models in point were first-order dynamic equations and Elovich equations, whereas parabolic diffusion equations were not suitable ones.

Adsorption↗

[The immunopotentiation of human B lymphocyte stimulator C-terminal peptide].

The cDNA of human B lymphocyte stimulator C-terminal peptide (C-BLyS) was amplified by nested PCR from cDNA library of human fetal brain. The expression plasmid pT7450-C-BLyS was constructed and transformed into E. coli BL21 CodonPlus (DE3) RIL which can recognize many rare codons. The C-BLyS protein was expressed as inclusion body in E. coli BL21 CodonPlus (DE3) RIL and the inclusion body of C-BLyS was denatured and then refolded by dialysis and purified by ion exchange chromatography. The refolded and purified C-BLyS can specifically bind with BCMA-Fc, a fusion protein of BLyS receptor and human IgG1 Fc. Furthermore, C-BLyS is proved to be an effective stimulator in mouse splenocytes proliferation in vitro and effective immunostimulant in vivo.

Animals↗

[RAPD analysis of genuiness on source of Bupleurum chinense].

OBJECTIVE: To identify the genuiness on 5 samples of Bupleurum chinense DC. from cultivating base in Dongfeng County, Jilin Province. METHODS: RAPD was used by 23 primers. RESULTS: The three primers can effectively identify the genuiness of Bupleurum chinense. CONCLUSION: The genuiness identification of Bupleurum chinense can be made with RAPD analysis.

Bupleurum↗

[The development and current situation of the methods for genetic evaluation in livestock].

Genetic evaluation is one of the most important components in livestock breeding program. Best linear unbiased prediction (BLUP) Animal Model, which based on phenotypic information,has become the most widely accepted method for genetic evaluation of domestic livestock. Large numbers of molecular genetic markers have been discovered as the development and application of genomics. Some new methods (Marked Assisted BLUP,MBLUP) for genetic evaluation have being developed,which incorporating molecular genetic markers information into genetic evaluation. MBULP will be a rapid and efficient method for genetic evaluation of domestic livestock. The aim of the paper is to introduce the development and current situation of the methods for livestock genetic evaluation.

English Abstract↗

Phosphorylation of Saccharomyces cerevisiae choline kinase on Ser30 and Ser85 by protein kinase A regulates phosphatidylcholine synthesis by the CDP-choline pathway.

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway.

Amino Acid Motifs↗

AC133-2, a novel isoform of human AC133 stem cell antigen.

Human AC133 antigen, also called CD133, was recently identified as a hematopoietic stem cell marker. However, the molecular structure and function of this protein has remained unclear. Here we cloned and identified a novel isoform of AC133, which we named AC133-2. In comparison to the reported AC133 cDNA, which is referred to herein as AC133-1, a small exon of 27 nucleotides is deleted in AC133-2 by alternative mRNA splicing. Similar to the previously characterized AC133 antigen, recombinant AC133-2 expressed in 293 cells was glycosylated and transported to plasma membrane. AC133-2 mRNA was found predominant in a variety of human fetal tissue, adult tissues, and several carcinomas. In contrast, AC133-1 mRNA was more prominent in fetal brain and adult skeletal muscle but was not detected in fetal liver and kidney, adult pancreas, kidney, and placenta, suggesting different roles for the two isoforms in fetal development and mature organ homeostasis. Here, we demonstrate that AC133-2 is the isoform expressed on hematopoietic stem cells derived from fetal liver, bone marrow, and peripheral blood. The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells. To further investigate its expression in other stem cell populations, we found that AC133-2 co-expressed with beta(1) integrin in the basal layer of human neonatal epidermis. AC133-2(+)/beta(1) integrin(+) cells proliferated and differentiated in culture, which coincided with a loss of AC133-2 and gain in a terminal differentiation marker involucrin. Taken together, these results suggest that AC133-2 is expressed in multiple stem cell niches and may provide a means to isolate specific stem cell subpopulations from human tissues.

AC133 Antigen↗

Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms.

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Valpha24+CD4+ NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-alpha cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-alpha killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-alpha mechanisms. TNF-alpha killing requires longer contact between effectors and targets, suggesting that TNF-alpha acts by enhancing perforin killing.

Antigens, CD1↗

Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation.

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+](cyt).

Blood Proteins↗

Role of capacitative Ca2+ entry in bronchial contraction and remodeling.

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+ in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents (I(SOC)) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+ channels by Ni2+ decreased I(SOC) and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I(SOC), enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC proliferation.

Acetylcholine↗