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1.
Org Biomol Chem ; 21(9): 1937-1941, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36752554

RESUMO

A guanidine group is abundantly found in natural products and drugs. Guanidine has the highest basicity among many common functional groups in nature. Because of its high basicity, it generally exists as a protonated guanidinium and functions as a cationic hydrogen bond donor. Finding an appropriate bioisostere of guanidinium is challenging because of its high basicity and unique trigonal planar shape. In this study, we explored the possibility of "deltic guanidinium" as a bioisostere of guanidinium using a cyclic arginine-glycine-aspartic acid (RGD) peptide as a parent compound. We synthesized c(deltic RGDyK), in which a guanidinium group of an arginine residue in c(RGDyK) is replaced with deltic guanidinium. A target binding assay, biodistribution study, and metabolic stability assay were conducted with c(deltic RGDyK) and its radioiodinated variant. The deltic guanidinium analog peptides exhibited similar biological properties to the parent peptides and improved in vivo stability, indicating that deltic guanidinium could work as a unique bioisostere of guanidinium.


Assuntos
Oligopeptídeos , Peptídeos , Guanidina/química , Distribuição Tecidual , Peptídeos/química , Oligopeptídeos/metabolismo , Arginina/química
2.
Jpn J Clin Oncol ; 50(12): 1395-1402, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32856068

RESUMO

OBJECTIVE: Idelalisib is an orally administered, highly selective inhibitor of phosphatidylinositol 3-kinase-δ. In this phase 1b study, the safety, tolerability and pharmacokinetics of idelalisib, an oral inhibitor of phosphatidylinositol 3-kinase-δ, were evaluated in Japanese patients with relapsed or refractory indolent B-cell non-Hodgkin lymphoma. METHODS: In total, six patients (follicular lymphoma: n = 3, chronic lymphocytic leukemia: n = 3) were enrolled to receive idelalisib 150 mg twice daily. RESULTS: No dose-limiting toxicities were reported. The most common adverse events were diarrhea (n = 5), gastritis (n = 3), insomnia (n = 3) and pyrexia (n = 3). The most common ≥grade 3 adverse events were diarrhea (n = 2), increased transaminase levels (n = 2) and decreased appetite (n = 2). The maximum idelalisib plasma concentrations (Cmax) were achieved at 2.50 h (range: 1.50-4.00 h). The mean idelalisib plasma concentrations decreased over time but remained detectable in most patients at 12 h. All enrolled patients underwent efficacy evaluation by investigators, and five patients (follicular lymphoma: n = 2, chronic lymphocytic leukemia: n = 3) achieved partial response. The median duration of partial response was 14.5 months (range: 3.7-31.3 months). CONCLUSION: Idelalisib 150 mg twice daily was considered tolerable in Japanese patients with follicular lymphoma or chronic lymphocytic leukemia.(Clinical trial registration: NCT02242045).


Assuntos
Antineoplásicos/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma Folicular/tratamento farmacológico , Purinas/administração & dosagem , Quinazolinonas/administração & dosagem , Administração Oral , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Esquema de Medicação , Humanos , Japão , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Purinas/efeitos adversos , Purinas/farmacocinética , Quinazolinonas/efeitos adversos , Quinazolinonas/farmacocinética , Recidiva , Segurança , Resultado do Tratamento
3.
ESC Heart Fail ; 11(2): 692-701, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38098210

RESUMO

AIMS: The trajectories of systolic function after admission for acute heart failure (HF) and their effect on clinical outcomes have not been fully elucidated. We aimed to assess changes in left ventricular ejection fraction (LVEF) between the index and 1 year after discharge and to examine their prognostic implications. METHODS AND RESULTS: We extracted data from a prospective multicentre registry of patients hospitalized for acute HF and identified 1636 patients with LVEF data at admission and 1 year after discharge. We categorized them into five groups based on LVEF changes: HF with unchanged-preserved EF [HFunc-pEF (EF ≥ 50%); N = 527, 32.2%], unchanged-mildly reduced EF [HFunc-mrEF (EF 41-49%); N = 86, 5.3%], unchanged-reduced EF [HFunc-rEF (EF ≤ 40%); N = 377, 23.0%], worsened EF (HFworEF; N = 83, 5.1%), and improved EF (HFimpEF; N = 563, 34.4%). We then evaluated the subsequent composite outcome of cardiovascular death and HF readmission. During 1 year after discharge, 53% of patients with HF with reduced EF and 67% of those with HF with mildly reduced EF (HFmrEF) transitioned to other categories, whereas 92% of those with HF with preserved EF (HFpEF) remained within the same category. Patients with HFimpEF were more likely to be younger and had relatively preserved renal function, whereas those with HFworEF were the oldest and had more comorbidities among the five groups. After multivariable adjustment, patients with HFimpEF and HFunc-pEF had a lower risk for composite outcomes when referenced to patients with HFunc-rEF [hazard ratio (95% confidence interval), P-value: 0.28 (0.16-0.49), P < 0.001, and 0.40 (0.25-0.63), P < 0.001, respectively]. Conversely, patients with HFunc-mrEF and HFworEF had a comparable risk [0.44 (0.18-1.07), P = 0.07, and 0.63 (0.29-1.39), P = 0.26, respectively]. CONCLUSIONS: A substantial number of patients with HF experienced transitions to other categories after discharge. Notably, patients with decreased EF experienced a worse prognosis, even with slight decreases (e.g. HFpEF transitioning to HFmrEF). These findings emphasize the significance of longitudinal assessments of systolic function to better manage patients following acute decompensation.


Assuntos
Insuficiência Cardíaca , Função Ventricular Esquerda , Humanos , Volume Sistólico , Estudos Prospectivos , Hospitalização
4.
Avian Dis ; 55(2): 187-94, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21793432

RESUMO

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).


Assuntos
Galinhas , Infecções por Mycoplasma/veterinária , Mycoplasma synoviae/genética , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/fisiologia , Genótipo , Japão/epidemiologia , Lectinas/genética , Lectinas/metabolismo , Dados de Sequência Molecular , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Filogenia , Doenças das Aves Domésticas/epidemiologia
5.
Monoclon Antib Immunodiagn Immunother ; 40(3): 124-133, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34042540

RESUMO

The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Leões-Marinhos/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetinae , Cricetulus , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/imunologia , Leões-Marinhos/genética
6.
Monoclon Antib Immunodiagn Immunother ; 38(2): 79-84, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30939066

RESUMO

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cavalos , Camundongos
7.
Monoclon Antib Immunodiagn Immunother ; 38(2): 89-95, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31009336

RESUMO

Podoplanin (PDPN), also known as T1alpha, has been used as a lung type I alveolar cell marker in the pathophysiological condition. Although we have established several monoclonal antibodies (mAbs) against mammalian PDPNs, mAbs against tiger PDPN (tigPDPN), which are useful for immunohistochemical analysis, remain to be developed. In this study, we immunized mice with tigPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/tigPDPN) and screened hybridomas producing mAbs against tigPDPN using flow cytometry. One of the mAbs, PMab-231 (IgG2a, kappa), specifically detected CHO/tigPDPN cells using flow cytometry as well as recognized tigPDPN protein using western blotting. In addition, PMab-231 was found to cross-react with cat PDPN (cPDPN). The dissociation constants (KD) of PMab-231 for CHO/tigPDPN and CHO/cPDPN cells were determined to be 1.2 × 10-8 and 1.9 × 10-8, respectively, indicating moderate affinity for CHO/tigPDPN and CHO/cPDPN cells. PMab-231 stained type I alveolar cells of the feline lungs and podocytes of the feline kidneys using immunohistochemistry. Our findings suggest the potential usefulness of PMab-231 for the functional analyses of tigPDPN and cPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Hibridomas/imunologia , Rim/metabolismo , Pulmão/metabolismo , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Células CHO , Gatos , Cricetinae , Cricetulus , Rim/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Tigres
8.
Heliyon ; 5(7): e02063, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31338471

RESUMO

Sensitive and specific monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb, PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/gPDPN) cells, and screened mAbs against gPDPN using flow cytometry. One of the mAbs, PMab-235 (IgG1, kappa), specifically detected CHO/gPDPN cells by flow cytometry. Furthermore, PMab-235 strongly detected lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. These findings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

9.
Biochem Biophys Rep ; 18: 100631, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30984883

RESUMO

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.

10.
Monoclon Antib Immunodiagn Immunother ; 38(3): 129-132, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31112076

RESUMO

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Tigres/metabolismo , Animais , Tigres/imunologia
11.
Monoclon Antib Immunodiagn Immunother ; 38(2): 96-99, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30457925

RESUMO

Podoplanin (PDPN) is expressed in type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. We have characterized the PDPNs of various animal species using specific anti-PDPN monoclonal antibodies (mAbs). In this study, we investigated whether these anti-PDPN mAbs cross-react with goat PDPN (gPDPN). Flow cytometry demonstrated that the anti-bovine PDPN mAb PMab-44 (IgG1, kappa) reacts with gPDPN, which is overexpressed in CHO-K1 cells. Using immunohistochemical analysis, type I alveolar cells of goat lung were strongly detected by PMab-44. These results indicate that PMab-44 is useful for investigating gPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Imuno-Histoquímica/métodos , Pulmão/metabolismo , Glicoproteínas de Membrana/imunologia , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Cabras , Pulmão/imunologia
12.
Monoclon Antib Immunodiagn Immunother ; 38(1): 18-24, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30802179

RESUMO

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos/genética
13.
Biochem Biophys Rep ; 18: 100616, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30766925

RESUMO

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.

14.
Monoclon Antib Immunodiagn Immunother ; 38(1): 30-36, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30681406

RESUMO

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos
15.
Monoclon Antib Immunodiagn Immunother ; 38(5): 213-219, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31403389

RESUMO

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Colo/imunologia , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/genética , Epitopos/metabolismo , Cabras , Imuno-Histoquímica , Pulmão/imunologia
16.
J Neurosci ; 27(39): 10520-9, 2007 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-17898223

RESUMO

The vesicular monoamine transporter 2 (VMAT2) is localized primarily within the CNS and is responsible for transporting monoamines from the cytoplasm into secretory vesicles. Because reserpine (a VMAT inhibitor) can precipitate depressive-like symptoms in humans, we investigated whether Vmat2 heterozygous (HET) mice present with depressive-like behaviors. The mutants showed locomotor and rearing retardation in the open field and appeared anhedonic to 1 and 1.5% sucrose solutions. Immobility times for Vmat2 heterozygotes were prolonged in forced swim and imipramine normalized this behavior. HET animals also showed enhanced immobility in tail suspension and this response was alleviated by fluoxetine, reboxetine, and bupropion. Stimulated GTPgammaS binding indicated that alpha2-adrenergic receptors in HET hippocampus were more sensitive to UK 14,304 (5-bromo-N-(4,5-dihydro-1-H-imidazol-2-yl)-6-quinoxalinamine) stimulation than in wild type (WT) mice. In learned helplessness, mice were exposed to a shuttle box for 4 d or were given inescapable foot-shocks for the same time period. On day 5, all animals were tested in shock escape. Failure rates and the latency to escape were similar for WT and HET mice that were only pre-exposed to the test apparatus. In foot-shock groups, learned helplessness was more robust in heterozygotes than in WT controls. Basal secretion of serum corticosterone was not distinguished by genotype; however, corticosterone levels in mutants were more responsive to stress. Anxiety-like responses of WT and HET animals in the open field, light-dark exploration, zero maze, and novelty-suppressed feeding tests were indistinguishable. Collectively, these findings suggest that Vmat2 heterozygotes display a depressive-like phenotype that is devoid of anxiety-like behavior.


Assuntos
Transtorno Depressivo Maior/genética , Proteínas Vesiculares de Transporte de Monoamina/genética , Animais , Antidepressivos/farmacologia , Ansiedade/genética , Corticosterona/biossíntese , Transtorno Depressivo Maior/diagnóstico , Modelos Animais de Doenças , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Testes Neuropsicológicos , Fenótipo
17.
Artigo em Inglês | MEDLINE | ID: mdl-30083132

RESUMO

Background and Aims: Hepatocyte growth factor (HGF) is a multifunctional pleiotropic protein involved in tissue regeneration, protection, angiogenesis, anti-inflammatory and anti-fibrotic responses, and tumorigenesis, through binding to its receptor MET. Recombinant HGF protein has been shown to mitigate various liver disease models, such as alcohol-induced liver injury, hepatic ischemia-reperfusion injury, and fibrosis. This study aimed to investigate the anti-inflammatory, anti-fibrotic, and anti-lipogenic effects of exogenous administration of feline HGF on a non-alcoholic steatohepatitis (NASH) mouse model. Methods: Wild-type C57BL/6 mice were fed a choline-deficient amino acid defined (CDAA) diet for 3 weeks to create the mouse model of NASH, which displays hepatic steatosis, inflammation, injury, and very mild fibrosis. One mg/kg of recombinant feline HGF was administered intravenously daily in the last 7 days of the total 3 weeks of CDAA diet feeding. Then, hepatic steatosis, inflammation, injury, and fibrogenic gene expression was examined. Results: After 3 weeks of a CDAA diet-feeding, the vehicle-treated mice exhibited evident deposition of lipid droplets in hepatocytes, inflammatory cell infiltration, and hepatocyte ballooning along with increased serum ALT levels whereas recombinant HGF-treated mice showed reduced hepatic steatosis, inflammation, and ballooned hepatocytes with a reduction of serum ALT levels. Recombinant HGF administration promoted hepatocyte proliferation. Increased hepatic lipid accumulation was accompanied by elevated expression of lipogenesis genes Fasn and Dgat1 in vehicle-treated mice. In HGF-treated mice, these genes were reduced with a decrease of lipid accumulation in the liver. Consistent with the anti-inflammatory property of HGF, augmented macrophage infiltration and upregulation of chemokines, Cxcl1, Ccl2, and Ccl5 in the CDAA diet fed mice, were suppressed by the addition of the HGF treatment. Finally, we examined the fibrotic response. The vehicle-treated mice had mild fibrosis with upregulation of Col1a1, Acta2, Timp1, Tgfb1, and Serpine1 expression. Recombinant HGF treatment significantly suppressed fibrogenic gene expression and collagen deposition in the liver. Conclusion: Recombinant feline HGF treatment suppressed the progression of NASH in a CDAA diet feeding mouse model.This suggests that recombinant HGF protein has therapeutic potential for NASH.

18.
Monoclon Antib Immunodiagn Immunother ; 37(4): 188-193, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30088964

RESUMO

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1-3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetinae , Cricetulus , Mapeamento de Epitopos , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Agregação Plaquetária/imunologia , Podócitos/imunologia , Ratos
19.
Monoclon Antib Immunodiagn Immunother ; 37(5): 233-237, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30362932

RESUMO

Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs, PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN (CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses. PMab-202 is expected to prove useful in investigating the function of horse PDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Especificidade de Anticorpos/imunologia , Células CHO , Cricetinae , Cricetulus , Mapeamento de Epitopos , Citometria de Fluxo , Cavalos/imunologia , Humanos , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Agregação Plaquetária/imunologia , Podócitos/imunologia
20.
Biochem Biophys Rep ; 15: 76-80, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30073207

RESUMO

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs.

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