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1.
J Pharmacol Exp Ther ; 370(1): 54-61, 2019 07.
Article in English | MEDLINE | ID: mdl-30992315

ABSTRACT

Endothelin receptor A (ETA) is a G protein-coupled receptor and a major therapeutic target for pulmonary arterial hypertension (PAH). We took a novel approach and developed an antagonistic monoclonal antibody, getagozumab, specifically against ETA. Getagozumab displayed a K d value of 8.7 nM and an IC50 value of 37.9 nM in the cell-based assays. Getagozumab could significantly lower pulmonary arterial pressure in both hypoxia-induced and monocrotaline (MCT)-induced PAH monkey models and further attenuate the pulmonary arterial and right ventricular hypertrophy in MCT-induced PAH monkeys. The preclinical studies demonstrated that getagozumab is safe, long lasting, and efficacious. Getagozumab may provide a new and effective treatment for PAH patients.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Pulmonary Arterial Hypertension/drug therapy , Pulmonary Arterial Hypertension/immunology , Receptor, Endothelin A/immunology , Animals , Antibodies, Monoclonal/pharmacokinetics , Cell Line , Female , Humans , Macaca fascicularis , Male , Pulmonary Arterial Hypertension/metabolism , Rats
2.
Biochem Pharmacol ; 150: 46-53, 2018 04.
Article in English | MEDLINE | ID: mdl-29355505

ABSTRACT

AIMS: Glucagon like-peptide-1 (GLP-1)-based drugs have been proposed as mono- or combined therapy for type 2 diabetes mellitus. Thus we characterized a novel antibody fusion protein engineered by linking the human GLP-1 derivative to a humanized GLP-1 receptor (GLP-1R) antibody via a peptide linker. MATERIALS AND METHODS: Glutazumab was characterized by receptor binding and reporter activation assays, and its specificity was investigated with the aid of the cognate receptor antagonist exendin (9-39) and antibody Ab1. Pharmacokinetics was evaluated in Sprague-Dawley (SD) rats and cynomolgus monkeys, and pharmacodynamics was assessed in normal ICR and spontaneous type 2 diabetic KKAy mice. Hypoglycemic effects were evaluated after acute administration and glucose metabolism and ß-cell function were assessed with repeated administrations. Dulaglutide was a positive control in all experiments. RESULTS: Glutazumab significantly bound and activated GLP-1R, but the receptor antagonist exendin (9-39) did not inhibit the activation except when combined with Ab1. Single injection of glutazumab reduced the blood glucose in ICR mice and KKAy mice, and the half-lives in SD rats and cynomolgus monkeys were 18 h and 33.6 h. Repeated injections of glutazumab controlled glycemic fluctuations and improved ß-cell function in KKAy mice. CONCLUSIONS: As a novel GLP-1R agonist, glutazumab may be a potential treatment for T2DM.


Subject(s)
Blood Glucose/metabolism , Drug Delivery Systems/methods , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/metabolism , Hypoglycemic Agents/metabolism , Animals , Binding Sites/drug effects , Binding Sites/physiology , Blood Glucose/drug effects , Female , Glucagon-Like Peptide 1/antagonists & inhibitors , Glucagon-Like Peptide-1 Receptor/antagonists & inhibitors , Humans , Hypoglycemic Agents/administration & dosage , Macaca fascicularis , Male , Mice , Mice, Inbred ICR , Mice, Transgenic , Rats , Rats, Sprague-Dawley
3.
Asian J Androl ; 12(4): 567-77, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20543856

ABSTRACT

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location of Dby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group (35.9% vs. 95%, P = 0.001) and the as-Dby female pronucleus injection group (35.9% vs. 93.8%, P = 0.001). The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group (17.4% vs. 57.9%, P = 0.002) and the as-Dby female pronucleus injection group (17.4% vs. 54.1%, P = 0.002). Thus, we conclude that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.


Subject(s)
DEAD-box RNA Helicases/genetics , Embryonic Development , RNA, Messenger/metabolism , Sperm Capacitation/genetics , Zygote/metabolism , Animals , Embryonic Development/drug effects , Female , Histone Demethylases , Male , Mice , Minor Histocompatibility Antigens , Pregnancy , Proteins/genetics , RNA, Antisense/pharmacology , Spermatozoa/metabolism , Testis/metabolism
4.
Int Immunopharmacol ; 10(4): 461-6, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20079464

ABSTRACT

Nicotine is a major component of cigarette smoking which may be involved in the progress of atherogenesis. In order to explain the mechanism of nicotine-induced endothelium dysfunction, we investigated the effects of nicotine on cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). Nicotine treatment increased the expressions of COX-2 at mRNA and protein level in a dose-dependent manner, following prostaglandin E(2) (PGE(2)) release enhancement. Pyrrolidine dithiocarbamate (PDTC, NF-kappaB inhibitor) and alpha-Bungarotoxin (alpha-Btx, nicotinic acetylcholine receptor antagonist) attenuated the nicotine-induced COX-2 expression and PGE(2) production. Furthermore, nicotine-induced ICAM-1 expression was reduced by NS-398 (selective COX-2 inhibitor). Taken together, the present study demonstrated that nicotine-induced COX-2 expression through NF-kappaB activation which mediated by nicotinic acetylcholine receptor and the induction of COX-2 was related to ICAM-1 expression.


Subject(s)
Cyclooxygenase 2/biosynthesis , Dinoprostone/biosynthesis , Endothelial Cells/enzymology , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Blotting, Western , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cells, Cultured , Electrophoretic Mobility Shift Assay , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme Induction/drug effects , Female , Fluorescent Antibody Technique , Humans , Indicators and Reagents , Intercellular Adhesion Molecule-1/biosynthesis , Nicotinic Antagonists/pharmacology , Pregnancy , Receptors, Nicotinic/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Stimulation, Chemical , Umbilical Veins/cytology
5.
Mol Reprod Dev ; 77(2): 158-66, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19834984

ABSTRACT

We asked if messenger ribonucleic acid (mRNA) of Y chromosome genes was selectively retained in human ejaculated spermatozoa. A panel of genes on the nonrecombining region of Y chromosome (NRY) and their X homologues were selected and screened in human testis and ejaculated spermatozoa by reverse transcription-polymerase chain reaction (RT-PCR). Then, the cellular localization of DBY, RPS4Y and SMCY mRNA in testis and matured spermatozoa were investigated by in situ hybridization. The expression pattern of these three genes in human ejaculated spermatozoa was also tested by Western blot and Immunofluorescence. All 11 Y chromosome gene mRNAs were found in the testis, but the transcripts of only three genes DBY, SRY, and RPS4Y were detected in ejaculated sperm by RT-PCR. Further investigation showed that DBY and RPS4Y mRNA were present in both human testis and the post-acrosome region comprising nearly 50% of spermatozoa, while the encoded proteins were not detected in spermatozoa. In contrast, although SMCY mRNA was not detected in mature spermatozoa, the H-Y antigen encoded by SMCY was expressed on the surface of spermatozoa. We therefore conclude that the spermatozoa mRNA could be selectively reserved in mature ejaculated spermatozoa. They might be useful in early zygotic and embryonic development.


Subject(s)
Chromosomes, Human, Y/metabolism , Gene Expression Regulation/physiology , RNA, Messenger/biosynthesis , Spermatozoa/metabolism , DEAD-box RNA Helicases/biosynthesis , Ejaculation , Humans , Male , Minor Histocompatibility Antigens , Spermatozoa/cytology
6.
Parasitol Res ; 105(6): 1539-48, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19690889

ABSTRACT

The role of heat shock protein 90 inhibitor geldanamycin (GA) during Leishmania donovani promastigote-to-amastigote transformation in axenic conditions was investigated. Promastigotes exhibited apoptotic morphologic changes after GA treatment at a high temperature, and the effect is in a dose- and time-dependant manner. Meanwhile, cell cycle analysis showed a significant increase at the expense of cells in the G0/G1 phase and a decrease in the S and G2/M phases after GA treatment. In addition, cellular glutathione level was reduced and reactive oxygen species content was increased afterwards. Pretreatment with antioxidants reduced the percentage of GA-induced cell apoptosis. After treatment, cultures in pH 5.5 showed a lower percentage of apoptosis than those in pH 7.4. The present study showed that GA could cause apoptosis in L. donovani but could not cause stage differentiation in high temperature and that acidic conditions were likely to be crucial for the transformation and survival of the parasite within its human host.


Subject(s)
Apoptosis , Benzoquinones/toxicity , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/toxicity , Leishmania donovani/drug effects , Leishmania donovani/growth & development , Animals , Antioxidants , Cell Cycle/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Glutathione/analysis , Hydrogen-Ion Concentration , Leishmania donovani/chemistry , Reactive Oxygen Species/analysis
7.
FEBS J ; 275(21): 5422-8, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18959766

ABSTRACT

Serial analysis of gene expression (SAGE) is a powerful technique for studying gene expression at the genome level. However, short SAGE tags limit the further study of related data. In this study, in order to identify a gene, we developed a semi-nested PCR-based method called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate longer 3'-end cDNA fragments from unknown SAGE tags. In the procedure, a modified lock-docking oligo(dT) with two degenerate nucleotide positions at the 3'-end was used as a reverse primer to synthesize cDNAs. Afterwards, the full-length cDNAs were amplified by PCR based on 5'-RACE and 3'-RACE. The amplified cDNAs were then used for the subsequent two-step PCR of the TSAT-PCR process. The first-step PCR was carried out at an appropriately low annealing temperature; a SAGE tag-specific primer was used as the sense primer, and an 18 bp sequence (universal primer I) located at the 5'-reverse primer end was used as the antisense primer. After 15-20 PCR cycles, the 3'-end cDNA fragments containing the tag could be enriched, and the PCR products could be used as templates for the second-step PCR to obtain the specific products. The second-step PCR was performed with a SAGE tag-specific primer and a 22-bp sequence (universal primer II) upstream of universal primer I at the 5'-reverse primer with a high annealing temperature. With our innovative TSAT-PCR method, we could easily obtain specific PCR products covering SAGE from those transcripts, especially low-abundance transcripts. It can be used as a method to identify genes expressed in different cell types.


Subject(s)
Gene Expression , Polymerase Chain Reaction/methods , DNA Primers , DNA, Complementary , Gene Expression Profiling/methods , RNA, Messenger/analysis , Sequence Analysis, DNA/methods
8.
Parasitol Res ; 103(4): 821-8, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18568446

ABSTRACT

In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) fetal bovine serum, were transferred from 26 degrees C to 37 degrees C. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively. Characteristics of the expressed genes' frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency>or=20), whose levels were equal to or >1.3% of the identified tags, constituted >23% of the total sequenced tags. Correspondingly, 75.72% or 71.65% of the genes accounted for those tags present at low abundance (frequency=1) contributed only 32.13% or 27.89% of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs.


Subject(s)
Gene Expression Profiling , Leishmania donovani/growth & development , Leishmania donovani/genetics , Animals , Expressed Sequence Tags , Gene Library , Genes, Protozoan , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Protozoan/biosynthesis , RNA, Protozoan/genetics , Temperature
9.
Int Immunopharmacol ; 6(6): 892-902, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16644474

ABSTRACT

Although gonadal hormone mostly causes genotropic actions through the members of nuclear receptor family, it also can regulate these actions via membrane receptor. To explore the possibility of plasma membrane estrogen receptors (mER) mediating genotropic events, we have investigated estrogen's effect on nicotine-stimulated adhesion molecule expression and evaluated whether this effect depends on calcium, MAPK signal pathway. Fluorescence Spectroscopy analysis of Ca2+ from human umbilical vein endothelial cells (HUVECs) showed through mER, estrogen induced a rapid rise of intracellular free Ca2+ concentration and this rise could not be inhibited by tamoxifen (classic ER inhibitor). In the context of nicotine stimulating, however, estrogen attenuated phosphorylation of mitogen-activated protein kinase (MAPK) family members, extracellular signal regulated kinase 1/2 (ERK1/2), p38 but not c-Jun-N-terminal kinase (JNK) in HUVECs and this effect could not still be prevented by tamoxifen. In the meantime, estrogen also down-regulated surface/soluble vascular cell adhesion molecule (VCAM-1, sVCAM-1) and endothelial selectin (E-selectin, sE-selectin) levels, which was not abolished by tamoxifen either. Moreover, calcium chelator BAPTA, ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 significantly reduced the production of nicotine-activated surface/soluble VCAM-1 and E-selectin and both of the remained levels were no longer regulated by estrogen. Our study here provides the information of decrease effect of mER-mediated estrogen through Ca2+ and ERK1/2, p38 MAPK signaling pathway on nicotine-stimulated expression of surface/soluble VCAM-1 and E-selectin in HUVECs.


Subject(s)
Cell Adhesion Molecules/metabolism , Endothelial Cells/drug effects , Estrogens/pharmacology , Nicotine/pharmacology , Calcium Signaling/drug effects , Cell Adhesion Molecules/genetics , Cells, Cultured , Chelating Agents/pharmacology , Down-Regulation/drug effects , E-Selectin/genetics , E-Selectin/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Endothelial Cells/chemistry , Endothelial Cells/metabolism , Estradiol/pharmacology , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression/drug effects , Humans , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Receptors, Estradiol/analysis , Tamoxifen/pharmacology , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
10.
Hum Reprod ; 21(6): 1583-90, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16501037

ABSTRACT

BACKGROUND: Accumulated evidence proves that mature spermatozoa contain a complex yet specific array of mRNA, which could provide information on the past events of spermatogenesis. OBJECTIVE: To quantitatively microdissect these mRNA transcripts by a digital approach. METHODS: Serial analysis of gene expression (SAGE) was used to study the mRNA transcripts from ejaculate of a fertile individual and of a pool of 10 fertile men. Online DAVID software suite was also utilized to cluster the UniGene data. RESULTS: A SAGE library from the individual produced 20,237 raw tags representing 2459 unique tags and that from pooled 10 men generated 21,052 raw tags representing 2712 unique tags. When the unique tags occurring > or = 4 times were analysed, 564 overlapping tags were produced by 638 unique tags from the individual and 682 from the pooled library. Fifty-four of these overlapped tags were considered to be novel genes. Online analysis of the overlapping tags revealed 25 functional gene groups, with the dominant one comprising 96 nuclear protein genes involving transcription and transcription regulation and also a group with 84 ribosomal subunit genes involving protein synthesis. CONCLUSION: A SAGE analysis of ejaculate from fertile men has revealed a large number of transcripts, which occur in steady frequencies and probably have important roles in spermatogenesis and fertilization.


Subject(s)
Fertility , Gene Expression Profiling/methods , RNA, Messenger/metabolism , Spermatozoa/metabolism , Adult , Chromosomes/ultrastructure , Gene Library , Humans , Male , RNA/metabolism , Transcription, Genetic
11.
Int J Biochem Cell Biol ; 38(2): 170-82, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16257255

ABSTRACT

To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.


Subject(s)
Calcium/metabolism , E-Selectin/metabolism , Endothelial Cells/metabolism , Intercellular Adhesion Molecule-1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nicotine/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Bungarotoxins/metabolism , Cells, Cultured , E-Selectin/genetics , Endothelial Cells/cytology , Endothelial Cells/drug effects , Enzyme Activation , Gene Expression Regulation , Humans , Intercellular Adhesion Molecule-1/genetics , MAP Kinase Signaling System/physiology , Mecamylamine/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Nicotine/pharmacology , Nicotinic Agonists/metabolism , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/metabolism , Vascular Cell Adhesion Molecule-1/genetics
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