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1.
iScience ; 25(11): 105324, 2022 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-36304121

RESUMEN

Efficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.

2.
BMC Cancer ; 21(1): 72, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33446132

RESUMEN

BACKGROUND: p-Boronophenylalanine (10BPA) is a powerful 10B drug used in current clinical trials of BNCT. For BNCT to be successful, a high (500 mg/kg) dose of 10BPA must be administered over a few hours. Here, we report BNCT efficacy after rapid, ultralow-dose administration of either tumor vasculature-specific annexin A1-targeting IFLLWQR (IF7)-conjugated 10BPA or borocaptate sodium (10BSH). METHODS: (1) IF7 conjugates of either 10B drugs intravenously injected into MBT2 bladder tumor-bearing mice and biodistribution of 10B in tumors and normal organs analyzed by prompt gamma-ray analysis. (2) Therapeutic effect of IF7-10B drug-mediated BNCT was assessed by either MBT2 bladder tumor bearing C3H/He mice and YTS-1 tumor bearing nude mice. RESULTS: Intravenous injection of IF7C conjugates of either 10B drugs into MBT2 bladder tumor-bearing mice promoted rapid 10B accumulation in tumor and suppressed tumor growth. Moreover, multiple treatments at ultralow (10-20 mg/kg) doses of IF7-10B drug-mediated BNCT significantly suppressed tumor growth in a mouse model of human YTS-1 bladder cancer, with increased Anxa1 expression in tumors and infiltration by CD8-positive lymphocytes. CONCLUSIONS: We conclude that IF7 serves as an efficient 10B delivery vehicle by targeting tumor tissues via the tumor vasculature and could serve as a relevant vehicle for BNCT drugs.


Asunto(s)
Anexina A1/metabolismo , Compuestos de Boro/administración & dosificación , Terapia por Captura de Neutrón de Boro/métodos , Neovascularización Patológica/radioterapia , Fragmentos de Péptidos/metabolismo , Fenilalanina/análogos & derivados , Neoplasias de la Vejiga Urinaria/radioterapia , Animales , Apoptosis , Compuestos de Boro/química , Compuestos de Boro/metabolismo , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Fenilalanina/administración & dosificación , Fenilalanina/química , Fenilalanina/metabolismo , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Ensayos Antitumor por Modelo de Xenoinjerto
3.
PLoS One ; 16(1): e0241157, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33406123

RESUMEN

We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.


Asunto(s)
Anexina A1/antagonistas & inhibidores , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Proteínas de Neoplasias/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Péptidos , Administración Oral , Animales , Anexina A1/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Br J Cancer ; 123(11): 1633-1643, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32921792

RESUMEN

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.


Asunto(s)
Anexina A1/metabolismo , Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas , Portadores de Fármacos/farmacología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Péptidos , Ratas
6.
J Histochem Cytochem ; 67(10): 759-770, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31246144

RESUMEN

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development.


Asunto(s)
Gastritis/prevención & control , Mucinas/fisiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Cruzamientos Genéticos , Mucosa Gástrica/química , Mucosa Gástrica/patología , Gastritis/genética , Gastritis/patología , Hiperplasia , Inflamación/genética , Ratones , Ratones Noqueados , Mucinas/deficiencia , ARN Mensajero/análisis , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Sulfotransferasas/deficiencia , Sulfotransferasas/genética , Sulfotransferasas/fisiología , Regulación hacia Arriba , Carbohidrato Sulfotransferasas
7.
Int J Urol ; 26(2): 284-290, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30506742

RESUMEN

OBJECTIVES: To evaluate the expression of annexin A1 protein in patients with renal cell carcinoma. METHODS: Annexin A1 expression was examined in renal cell carcinoma specimens from 27 patients, and their disease-free survival was analyzed using the log-rank test. Annexin A1 knockdown in the human renal cell carcinoma cell line Caki-1 was carried out, and its proliferation, invasion, motility and adhesion were compared with those of control cells. RESULTS: In 13 out of 27 patients, annexin A1 was highly expressed in the membrane of renal cell carcinoma tumor cells, whereas in the rest of the patients, annexin A1 expression was weak or negligible in the membrane of those cells. Patients with high annexin A1 expression had significantly poorer disease-free survival than those with weak or negligible annexin A1 expression (P = 0.031). In the renal cell carcinoma cell line, annexin A1 knockdown cells showed significantly decreased proliferation, invasion, motility and adhesion relative to control cells, and expressed lower relative levels of membrane-type 1 matrix metalloproteinase and hypoxia-inducible factor 1-alpha transcripts, showing a potential pathway regulated by annexin A1. CONCLUSION: Annexin A1 is associated with renal cell carcinoma malignant potential and could serve as a marker of poor prognosis.


Asunto(s)
Anexina A1/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Anciano , Anciano de 80 o más Años , Anexina A1/genética , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/mortalidad , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/mortalidad , Masculino , Metaloproteinasa 14 de la Matriz/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica/patología , Pronóstico , Análisis de Supervivencia
8.
Sci Signal ; 8(406): ra124, 2015 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-26645581

RESUMEN

Melanoma is one of the most lethal skin cancers worldwide, primarily because of its propensity to metastasize. Thus, the elucidation of mechanisms that govern metastatic propensity is urgently needed. We found that protein kinase Cε (PKCε)-mediated activation of activating transcription factor 2 (ATF2) controls the migratory and invasive behaviors of melanoma cells. PKCε-dependent phosphorylation of ATF2 promoted its transcriptional repression of the gene encoding fucokinase (FUK), which mediates the fucose salvage pathway and thus global cellular protein fucosylation. In primary melanocytes and cell lines representing early-stage melanoma, the abundance of PKCε-phosphorylated ATF2 was low, thereby enabling the expression of FUK and cellular protein fucosylation, which promoted cellular adhesion and reduced motility. In contrast, increased expression of the gene encoding PKCε and abundance of phosphorylated, transcriptionally active ATF2 were observed in advanced-stage melanomas and correlated with decreased FUK expression, decreased cellular protein fucosylation, attenuated cell adhesion, and increased cell motility. Restoring fucosylation in mice either by dietary fucose supplementation or by genetic manipulation of murine Fuk expression attenuated primary melanoma growth, increased the number of intratumoral natural killer cells, and decreased distal metastasis in murine isograft models. Tumor microarray analysis of human melanoma specimens confirmed reduced fucosylation in metastatic tumors and a better prognosis for primary melanomas that had high abundance of fucosylation. Thus, inhibiting PKCε or ATF2 or increasing protein fucosylation in tumor cells may improve clinical outcome in melanoma patients.


Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Fucosa/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Transcripción Activador 2/genética , Animales , Línea Celular Tumoral , Fucosa/genética , Glicosilación , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética
9.
J Cell Biochem ; 116(6): 954-68, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25560148

RESUMEN

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) regulate a wide range of cellular processes. KRAB-ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB-ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777-mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777-mediated down-regulation of FAM129A induces moderate levels of the cyclin-dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777-mediated down-regulation of FAM129A plays a role in inhibition of cell proliferation.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Interferencia de ARN , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética
10.
J Biol Chem ; 289(23): 16462-77, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24753245

RESUMEN

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.


Asunto(s)
Antígenos de Neoplasias/química , Mucinas/química , Neoplasias de la Próstata/inmunología , Secuencia de Carbohidratos , Humanos , Masculino , Datos de Secuencia Molecular
11.
J Biol Chem ; 289(23): 16478-86, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24753248

RESUMEN

This study reports the determination of the carbohydrate epitope of monoclonal antibody F77 previously raised against human prostate cancer PC-3 cells (Zhang, G., Zhang, H., Wang, Q., Lal, P., Carroll, A. M., de la Llera-Moya, M., Xu, X., and Greene, M. I. (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 732-737). We performed a series of co-transfections using mammalian expression vectors encoding specific glycosyltransferases. We thereby identified branching enzymes and FUT1 (required for Fucα1→2Gal linkage) as being essential for F77 antigen formation. When immortalized normal prostate 267B1 cells were transfected with FUT1 alone, cells showed weak expression of F77 antigen. By contrast, cells co-transfected with FUT1 plus either GCNT1, GCNT2, or GCNT3 (an enzyme required to form GlcNAcß1→6Gal/GalNAc) showed robust F77 antigen expression, suggesting that F77 specifically binds to Fucα1→2Galß1→4GlcNAcß1→6Gal/GalNAc. RT-PCR for FUT1, GCNT1, GCNT2, and GCNT3 showed that F77-positive cell lines indeed express transcripts encoding FUT1 plus one GCNT. F77-positive prostate cancer cells transfected with siRNAs targeting FUT1, GCNT2, and GCNT3 showed significantly reduced F77 antigen, confirming the requirement of these enzymes for epitope synthesis. We also found that hypoxia induces F77 epitope expression in immortalized prostate RWPE1 cells, which express F77 antigen moderately under normoxia but at an elevated level under hypoxia. Quantitative RT-PCR demonstrated up-regulation of FUT1, GCNT2, and GCNT3 transcripts in RWPE1 cells under hypoxia, suggesting that hypoxia up-regulates glycosyltransferase expression required for F77 antigen synthesis. These results define the F77 epitope and provide a potential mechanism for F77 antigen synthesis in malignant prostate cancer.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Glicosiltransferasas/genética , Antígeno Prostático Específico/inmunología , Sistema del Grupo Sanguíneo ABO/inmunología , Secuencia de Bases , Secuencia de Carbohidratos , Línea Celular Tumoral , Cartilla de ADN , Humanos
12.
FEBS Lett ; 587(19): 3195-201, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-23968720

RESUMEN

Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-ß-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells.


Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Polisacáridos/fisiología , Transducción de Señal/fisiología , Glicósido Hidrolasas/farmacología , Células HEK293 , Humanos , Masculino , Polisacáridos/metabolismo , Unión Proteica , Motilidad Espermática/efectos de los fármacos , Espermatozoides/metabolismo
13.
Methods Mol Biol ; 1022: 369-86, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23765676

RESUMEN

Annexin A1 (Anxa1) is a highly specific surface marker of tumor vasculature. We used peptide-displaying phage technology to identify a carbohydrate ligand-mimicking 7-mer peptide, IFLLWQR (IF7), which can target Anxa1 in tumor vasculature. Here, we describe the binding activity of carbohydrate to Anxa1, Anxa1 to heparan sulfates, and the therapeutic potential of IF7 conjugated with anticancer drugs in tumor targeting.


Asunto(s)
Anexina A1/metabolismo , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Línea Celular Tumoral , Femenino , Colorantes Fluorescentes/química , Heparitina Sulfato/metabolismo , Humanos , Ratones , Neoplasias/irrigación sanguínea , Péptidos/química , Unión Proteica
14.
Transplantation ; 95(3): 418-25, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23274971

RESUMEN

BACKGROUND: Antibody-mediated rejection after ABO-incompatible kidney transplantation (ABO-I KTx) is a major barrier to transplantation success. The advent of immunosuppressive therapy has markedly improved graft survival in ABO-I KTx. However, compared with normal KTx, clinical conditions during ABO-I KTx are difficult to control because of overimmunosuppression. To reduce the need for immunosuppression, we aimed to develop a novel blood group antigen-neutralizing therapy. METHODS: We screened for an ABO blood group antigen-targeting peptide (BATP) by screening of T7 phage-displayed peptide library. After screening, hemagglutination inhibition assays, enzyme-linked immunosorbent assay, and cytotoxicity assay were used to analyze the blood group antigen-blocking effect and toxicity of BATP. We also tested the inhibitory effects on anti-blood group antibody binding in normal human kidney tissues blocked with BATP and excised kidneys perfused ex vivo with BATP. RESULTS: We identified six peptide sequences that efficiently suppressed hemagglutination of red blood cells by anti-ABO blood group antibodies and binding of these antibodies to ABO histo-blood group antigens in kidney tissues. Surprisingly, ex vivo perfusion of BATP in kidneys excised from renal cell carcinoma patients caused significant suppression of anti-blood group antibody binding to antigen and IgG and IgM deposition in renal glomerular capillaries after ABO-I blood reperfusion. CONCLUSIONS: These data indicate that A/B blood group antigens on red blood cells and in kidney tissues may be neutralized by BATP. This approach may enable the development of a novel blood group antigen-neutralizing therapy to overcome the challenges of ABO-I KTx.


Asunto(s)
Anticuerpos Antiidiotipos/fisiología , Antígenos de Grupos Sanguíneos/inmunología , Incompatibilidad de Grupos Sanguíneos/inmunología , Capilares/inmunología , Glomérulos Renales/irrigación sanguínea , Trasplante de Riñón/inmunología , Péptidos/fisiología , Capilares/patología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Humanos , Inmunoglobulina G/fisiología , Inmunoglobulina M/fisiología , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Reperfusión
15.
J Biol Chem ; 288(7): 5007-16, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23269668

RESUMEN

Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10(-/-) mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10(-/-) females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10(-/-) females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.


Asunto(s)
Esteroides/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Animales , Estrógenos/sangre , Femenino , Regulación de la Expresión Génica , Vectores Genéticos , Ácido Glucurónico/química , Glucolípidos/metabolismo , Células HEK293 , Humanos , Células Asesinas Naturales/citología , Ratones , Ratones Transgénicos , Modelos Genéticos , Neuronas/metabolismo , Recombinación Genética , Testosterona/sangre
16.
Glycoconj J ; 30(2): 183-94, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22653491

RESUMEN

In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcß1-4GlcNAcß1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MS(n), we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.


Asunto(s)
Lactosa/análogos & derivados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Bovinos , Lactosa/química , Tirotropina/química
17.
Reprod Biol Endocrinol ; 10: 101, 2012 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-23194061

RESUMEN

BACKGROUND: Trophinin is an intrinsic membrane protein that forms a complex in the cytoplasm with bystin and tastin, linking it microtubule-associated motor dynein (ATPase) in some cell types. Previously, we found that human sperm tails contain trophinin, bystin and tastin proteins, and that trophinin-binding GWRQ (glycine, tryptophan, arginine, glutamine) peptide enhanced motility of human sperm. METHODS: Immunohistochemistry was employed to determine trophinin protein in mouse spermatozoa from wild type mouse, by using spermatozoa from trophinin null mutant mice as a negative control. Multivalent 8-branched GWRQ (glycine, tryptophan, arginine, glutamine) peptide or GWRQ-MAPS, was chemically synthesized, purified by HPLC and its structure was confirmed by MALDI-TOF mass spectrometry. Effect of GWRQ-MAPS on mouse spermatozoa from wild type and trophinin null mutant was assessed by a computer-assisted semen analyzer (CASA). RESULTS: Anti-trophinin antibody stained the principal (central) piece of the tail of wild type mouse sperm, whereas the antibody showed no staining on trophinin null sperm. Phage particles displaying GWRQ bound to the principal piece of sperm tail from wild type but not trophinin null mice. GWRQ-MAPS enhanced motility of spermatozoa from wild type but not trophinin null mice. CASA showed that GWRQ-MAPS enhanced both progressive motility and rapid motility in wild type mouse sperm. CONCLUSIONS: Present study established the expression of trophinin in the mouse sperm tail and trophinin-dependent effect of GWRQ-MAPS on sperm motility. GWRQ causes a significant increase in sperm motility.


Asunto(s)
Moléculas de Adhesión Celular/fisiología , Péptidos/fisiología , Motilidad Espermática/fisiología , Regulación hacia Arriba/fisiología , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/química , Péptidos/metabolismo , Unión Proteica/fisiología
18.
Biol Pharm Bull ; 35(10): 1626-32, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23037151

RESUMEN

Although numerous carbohydrates play significant roles in mammalian cells, development of carbohydrate-based reagents and therapeutics are hampered by the technical difficulty of chemically synthesizing complex carbohydrate structures. Use of carbohydrate mimetic peptides circumvents this difficulty, as short peptide can be easily synthesized and modified. We as well as others identified carbohydrate mimetic peptides by screening peptide displaying phage library using anti-carbohydrate antibodies and lectins. This review introduces our experiences with I-peptide that was used for identification of new carbohydrate binding receptor expressed in the lung endothelial cells, and those with IF7 peptide that can be used as a therapeutic against malignant tumors.


Asunto(s)
Neoplasias/metabolismo , Péptidos/farmacología , Animales , Anexina A1/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Carbohidratos , Humanos , Pulmón/metabolismo , Neoplasias/tratamiento farmacológico , Péptidos/química , Péptidos/uso terapéutico
20.
Sheng Li Xue Bao ; 64(3): 247-58, 2012 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-22717627

RESUMEN

The process of human embryo implantation is mediated not only by evolutionarily conserved mechanisms, but also by a mechanism unique to humans. Evidence suggests that the cell adhesion molecules, L-selectin and trophinin, play a unique role in human embryo implantation. Here, we describe the dual roles of mucin carbohydrate ligand for L-selectin and trophinin protein and of the trophinin-associated proteins bystin and tastin. We then describe trophinin-mediated signal transduction in trophectoderm cells and endometrial epithelial cells. This review also covers cadherin and integrin in human embryo implantation.


Asunto(s)
Moléculas de Adhesión Celular/fisiología , Implantación del Embrión , Células Epiteliales/metabolismo , Transducción de Señal , Cadherinas/fisiología , Humanos , Integrinas/fisiología , Selectina L/fisiología
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