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2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(6): 1812-1819, 2019 Dec.
Artículo en Chino | MEDLINE | ID: mdl-31839043

RESUMEN

OBJECTIVE: To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1). METHODS: The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group. RESULTS: The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to ß- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P<0.05). CONCLUSION: The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.


Asunto(s)
Eucariontes , Glicoproteínas/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Transfección
3.
J Agric Food Chem ; 67(50): 13922-13928, 2019 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-31746600

RESUMEN

We compared phospholipids (PLs), PL fatty acid (FA) composition, and milk fat globule size and structure in human milk (n = 120) from mothers of full-term and preterm infants during lactation (colostrum, transition, 1 mo, 2 mo, and 3 mo) and 8 brands of infant formulas. The absolute quantification of PLs was analyzed using 31P NMR spectroscopy. Sphingomyelin was the dominant PLs (35.01 ± 3.31%) in human milk, whereas phosphatidylcholine and phosphatidylethanolamine were the dominant PLs in infant formulas. The PL content in preterm milk increased during lactation, whereas that in term milk remained stable. Saturated FAs (mainly 16:0 and 18:0) were the most abundant (>60%) PL FA in both preterm and term milk and increased throughout lactation. The mean diameter of milk fat globules in infant formulas was much smaller than that found in human milk (200 nm vs 5.63 µm). Significant differences were observed between human milk and infant formulas with regard to PLs, suggesting that more research is needed to mimic the PL profile in infant formulas.


Asunto(s)
Glucolípidos/química , Glicoproteínas/química , Fórmulas Infantiles/química , Leche Humana/química , Fosfolípidos/química , Embarazo/metabolismo , Adulto , Femenino , Edad Gestacional , Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Humanos , Lactante , Lactancia , Masculino , Leche Humana/metabolismo , Fosfolípidos/metabolismo
4.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 44(9): 976-984, 2019 Sep 28.
Artículo en Chino | MEDLINE | ID: mdl-31645485

RESUMEN

OBJECTIVE: To explore the effects of miR-101-3p on IL-1ß-induced chondrocyte injury and its underlying mechanisms.
 Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1ß group, a negative control group (IL-1ß+miR-NC group), and a miR-101-3p group (IL-1ß+miR-101-3p group), which were treated with IL-1ß after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1ß (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1ß+miR-101-3p group), an over-expression control group (IL-1ß+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1ß+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II.
 Results: Compared with the 0 ng/mL IL-1ß, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1ß (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1ß group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1ß group compared with the NC group (both P<0.05). Compared with the IL-1ß+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1ß+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1ß+miR-101-3p group compared with the IL-1ß+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1ß-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes.
 Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1ß-induced chondrocyte injury.


Asunto(s)
Condrocitos , Glicoproteínas/metabolismo , Interleucina-1beta/metabolismo , MicroARNs , Proliferación Celular
5.
Virchows Arch ; 475(6): 687-692, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31578606

RESUMEN

The 2017 World Health Organization (WHO) classification proposes to type and subtype primary adenohypophyseal tumours according to their cell lineages with the aim to establish more uniform tumour groups. The definition of atypical adenoma was removed in favour of high-risk adenoma, and the assessment of proliferative activity and invasion was recommended to diagnose aggressive tumours. Recently, the International Pituitary Pathology Club proposed to replace adenoma with the term of pituitary neuroendocrine tumour (PitNET) to better reflect the similarities between adenohypophyseal and neuroendocrine tumours of other organs. The European Pituitary Pathology Group (EPPG) endorses this terminology and develops practical recommendations for standardised reports of PitNETs that are addressed to histo- and neuropathologists. This brief report presents the results of EPPG's consensus for the reporting of PitNETs and proposes a diagnostic algorithm.


Asunto(s)
Glucosiltransferasas/metabolismo , Glicoproteínas/metabolismo , Tumores Neuroendocrinos/diagnóstico , Neoplasias Hipofisarias/diagnóstico , Neoplasias Hipofisarias/patología , Consenso , Humanos , Tumores Neuroendocrinos/patología , Sistemas Neurosecretores/patología , Organización Mundial de la Salud
6.
EMBO J ; 38(22): e101603, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31566781

RESUMEN

Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll ß-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.


Asunto(s)
Empalme Alternativo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Laminina/metabolismo , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuropéptidos/química , Neuropéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , Cristalografía por Rayos X , Glicoproteínas/genética , Ligandos , Ratones , Modelos Moleculares , Moléculas de Adhesión de Célula Nerviosa/genética , Neuropéptidos/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ratas , Homología de Secuencia
7.
J Agric Food Chem ; 67(42): 11675-11683, 2019 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-31545598

RESUMEN

To better appreciate the alterations of egg proteins and their modifications during embryonic development, a comparative and quantitative study was performed aimed at chicken egg white and yolk proteome and N-glycoproteome after 12 days of incubation using tandem mass tag (TMT)-labeling technology in conjunction with reversed-phase high-performance liquid chromatography (RP-HPLC). A total of 334 unique N-glycosite-containing peptides from 153 N-glycoproteins were identified, of which 82 N-glycosite-containing peptides showed significant changes after 12 days of incubation. The varied proteome was mainly involved with antibacterial, ionic binding, cell proliferation, and embryonic development, while the different degrading and/or absorbing priorities of egg proteins were proposed. This study provides substantial insight into the effects of N-glycoprotein variations on the utilization of egg proteins by chicken embryo during incubation.


Asunto(s)
Embrión de Pollo/química , Proteínas del Huevo/química , Glicoproteínas/química , Animales , Embrión de Pollo/crecimiento & desarrollo , Embrión de Pollo/metabolismo , Pollos/metabolismo , Cromatografía Líquida de Alta Presión , Proteínas del Huevo/metabolismo , Clara de Huevo/química , Yema de Huevo/química , Yema de Huevo/metabolismo , Glicoproteínas/metabolismo , Proteómica , Espectrometría de Masas en Tándem
8.
J Agric Food Chem ; 67(38): 10702-10712, 2019 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-31490688

RESUMEN

Human milk oligosaccharides are complex carbohydrates with multibiofunctional health benefits to newborns. Human milk free oligosaccharides (HMOs) are well characterized. However, changes in the N/O-glycome during lactation are poorly reported. Herein, we qualitatively and quantitatively investigated N/O-glycome profiles and their alteration in human milk at different lactation stages. N-Glycans were mainly fucosylated and nonsialylated, nonfucosylated throughout lactation. O-Glycans mainly consisted of sialylated and nonsialylated, nonfucosylated in colostrum and transitional milk, and fucosylated and nonfucosylated, nonsialylated in mature milk. Fucosylated and sialylated N-glycans gradually decreased and increased, respectively, as lactation progressed; O-glycans showed the reverse. Interestingly, changes in HMO abundance decreased during lactation, complementing HMG N/O-glycome changes. In conclusion, temporal HMG glycosylation changes provide the groundwork for developing infant formula that is closer to breast milk at different lactation stages.


Asunto(s)
Glicoproteínas/química , Lactancia , Leche Humana/química , Adulto , Calostro/química , Femenino , Glicoproteínas/metabolismo , Glicosilación , Humanos , Espectrometría de Masas , Leche Humana/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo
9.
J Agric Food Chem ; 67(35): 9950-9957, 2019 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-31403788

RESUMEN

Protein glycosylation is a ubiquitous posttranslational modification that modulates protein properties, thereby influencing bioactivities within a system. Duck egg white (DEW) proteins exhibit diverse biological properties compared with their chicken egg white (CEW) counterparts, which might be related to glycosylation. N-Glycoproteome analysis of DEW was conducted, and a total of 231 N-glycosites from 68 N-glycoproteins were identified. Gene ontology analysis was used to elucidate the biofunctions of DEW N-glycoproteins and compare them with those of CEW, which showed that the differences mostly involved molecular functions and biological processes. The biological functions of DEW N-glycoproteins were illuminated through bioinformatics analysis and comparison with CEW orthologues, which showed different allergenicities and antibacterial abilities. These divergences might be initiated by specific alterations in glycosylation, which can enhance the proteolysis resistance and protein steric hindrance. These results provide new insights for discovering the effects of N-glycosylation on biofunctions during the divergence of homologous proteins.


Asunto(s)
Pollos/genética , Patos/genética , Proteínas del Huevo/química , Glicoproteínas/química , Animales , Evolución Biológica , Pollos/metabolismo , Patos/metabolismo , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Clara de Huevo/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Proteómica
10.
Planta ; 250(5): 1703-1715, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31414205

RESUMEN

MAIN CONCLUSION: The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, ß-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30 °C for up to 40 h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion.


Asunto(s)
Néctar de las Plantas/metabolismo , Proteoma , Proteómica , Tabaco/enzimología , Electroforesis en Gel Bidimensional , Flores/genética , Flores/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Néctar de las Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/genética
11.
J Biotechnol ; 304: 63-69, 2019 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-31442500

RESUMEN

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.


Asunto(s)
Glicoproteínas/genética , Glicoproteínas/metabolismo , Virus de la Rabia/metabolismo , Virus de los Bosques Semliki/genética , Animales , Línea Celular , Electroporación , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Ingeniería de Proteínas , Virus de la Rabia/genética , Transfección , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo
12.
Asian Pac J Cancer Prev ; 20(8): 2281-2285, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31450896

RESUMEN

Introduction: Lung cancer is the most common cause of cancer-related death among males and females. The diagnosis of lung cancer is of great importance for clinical considerations and follow-up treatment. This study aimed to examine the expression of CEA, LUNX, and CK19 biomarkers in the cancerous and healthy tissues of patients suffering from NSCLC. Methods: In this study, 30 patients with NSCLCs referring to Masih Daneshvari Hospital in Tehran were voluntarily selected prior to taking any treatment. A tissue sample from the center and a sample of healthy tissues close to the cancerous masses were prepared by a specialist in the bronchoscopy sector and tested using real-time RT-PCR. Results: Positive CEA mRNA was observed in cancerous tissues in the center of tumors of 25 out of 30 cases. In the healthy tissue group, the same was found in 10 out of 30 cases (P<0.001). The markers CK19 and LUNX mRNAs showed to be positive in cancerous samples in the center of tumors of 15 and 22 out of 30 cases, and in the healthy tissue group, the expression was observed in 5 and 4 out of 30 cases, respectively(P<0.001). Conclusion: This study confirms that the aformentioed markers are the ones with a relatively appropriate sensitivity and specificity for the diagnosis of lung cancer.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Antígeno Carcinoembrionario/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Glicoproteínas/metabolismo , Queratina-19/metabolismo , Neoplasias Pulmonares/patología , Fosfoproteínas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico
13.
Adv Virus Res ; 104: 123-146, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31439147

RESUMEN

Fusion of viral and cellular membranes is an essential step in the entry pathway of all enveloped viruses. This is a dynamic and multistep process, which has been extensively studied, resulting in the endpoints of the reaction being firmly established, and many essential cellular factors identified. What remains is to elucidate the dynamic events that underlie this process, including the order and timing of glycoprotein conformational changes, receptor-binding events, and movement of the glycoprotein on the surface of the virion. Due to the inherently asynchronous nature of these dynamics, there has been an increased focus on the study of single virions and single molecules. These techniques provide researchers the high precision and resolution necessary to bridge the gaps in our understanding of viral membrane fusion. This review highlights the advancement of single-molecule and single-particle fluorescence-based techniques, with a specific focus on how these techniques have been used to study the dynamic nature of the viral fusion pathway.


Asunto(s)
Glicoproteínas/metabolismo , Fusión de Membrana , Imagen Óptica/métodos , Proteínas del Envoltorio Viral/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Microscopía/métodos
14.
Adv Virus Res ; 104: 147-183, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31439148

RESUMEN

Rhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus.


Asunto(s)
Interacciones Huésped-Patógeno , Virus de la Rabia/fisiología , Vesiculovirus/fisiología , Acoplamiento Viral , Internalización del Virus , Endocitosis , Glicoproteínas/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo
15.
Adv Virus Res ; 104: 225-281, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31439150

RESUMEN

Membrane fusion is a fundamental biological process that allows different cellular compartments delimited by a lipid membrane to release or exchange their respective contents. Similarly, enveloped viruses such as alphaherpesviruses exploit membrane fusion to enter and infect their host cells. For infectious entry the prototypic human Herpes simplex viruses 1 and 2 (HSV-1 and -2, collectively termed HSVs) and the porcine Pseudorabies virus (PrV) utilize four different essential envelope glycoproteins (g): the bona fide fusion protein gB and the regulatory heterodimeric gH/gL complex that constitute the "core fusion machinery" conserved in all members of the Herpesviridae; and the subfamily specific receptor binding protein gD. These four components mediate attachment and fusion of the virion envelope with the host cell plasma membrane through a tightly regulated sequential activation process. Although PrV and the HSVs are closely related and employ the same set of glycoproteins for entry, they show remarkable differences in the requirements for fusion. Whereas the HSVs strictly require all four components for membrane fusion, PrV can mediate cell-cell fusion without gD. Moreover, in contrast to the HSVs, PrV provides a unique opportunity for reversion analyses of gL-negative mutants by serial cell culture passaging, due to a limited cell-cell spread capacity of gL-negative PrV not observed in the HSVs. This allows a more direct analysis of the function of gH/gL during membrane fusion. Unraveling the molecular mechanism of herpesvirus fusion has been a goal of fundamental research for years, and yet important mechanistic details remain to be uncovered. Nevertheless, the elucidation of the crystal structures of all key players involved in PrV and HSV membrane fusion, coupled with a wealth of functional data, has shed some light on this complex puzzle. In this review, we summarize and discuss the contemporary knowledge on the molecular mechanism of entry and membrane fusion utilized by the alphaherpesvirus PrV, and highlight similarities but also remarkable differences in the requirements for fusion between PrV and the HSVs.


Asunto(s)
Herpesvirus Humano 1/fisiología , Herpesvirus Suido 1/fisiología , Herpesvirus Humano 2/fisiología , Internalización del Virus , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Acoplamiento Viral
16.
Adv Virus Res ; 104: 283-312, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31439151

RESUMEN

In this chapter, we present an overview on betaherpesvirus entry, with a focus on human cytomegalovirus, human herpesvirus 6A and human herpesvirus 6B. Human cytomegalovirus (HCMV) is a complex human pathogen with a genome of 235kb encoding more than 200 genes. It infects a broad range of cell types by switching its viral ligand on the virion, using the trimer gH/gL/gO for infection of fibroblasts and the pentamer gH/gL/UL128/UL130/UL131 for infection of other cells such as epithelial and endothelial cells, leading to membrane fusion mediated by the fusion protein gB. Adding to this scenario, however, accumulating data reveal the actual complexity in the viral entry process of HCMV with an intricate interplay among viral and host factors. Key novel findings include the identification of entry receptors platelet-derived growth factor-α receptor (PDGFRα) and Netropilin-2 (Nrp2) for trimer and pentamer, respectively, the determination of atomic structures of the fusion protein gB and the pentamer, and the in situ visualization of the state and arrangement of functional glycoproteins on virion. This is covered in the first part of this review. The second part focusses on HHV-6 which is a T lymphotropic virus categorized as two distinct virus species, HHV-6A and HHV-6B based on differences in epidemiological, biological, and immunological aspects, although homology of their entire genome sequences is nearly 90%. HHV-6B is a causative agent of exanthema subitum (ES), but the role of HHV-6A is unknown. HHV-6B reactivation occasionally causes encephalitis in patients with hematopoietic stem cell transplant. The HHV-6 specific envelope glycoprotein complex, gH/gL/gQ1/gQ2 is a viral ligand for the entry receptor. Recently, each virus has been found to recognize a different cellular receptor, CD46 for HHV 6A amd CD134 for HHV 6B. These findings show that distinct receptor recognition differing between both viruses could explain their different pathogenesis.


Asunto(s)
Citomegalovirus/fisiología , Herpesvirus Humano 6/fisiología , Internalización del Virus , Células Endoteliales/virología , Células Epiteliales/virología , Fibroblastos/virología , Glicoproteínas/metabolismo , Humanos , Proteína Cofactora de Membrana/metabolismo , Neuropilina-2/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores OX40/metabolismo , Receptores Virales/metabolismo , Linfocitos T/virología , Proteínas del Envoltorio Viral/metabolismo
17.
Cell Prolif ; 52(5): e12651, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31297902

RESUMEN

OBJECTIVE: It is essential to characterize underlying molecular mechanism associated with head and neck squamous cell carcinoma (HNSCC) and identify promising therapeutic targets. Herein, we explored role of homeobox transcript antisense RNA (HOTAIR) in HNSCC to regulate stanniocalcin-2 (STC2) by sponging microRNA-206 (miR-206). METHODS: HNSCC-related differentially expressed genes and regulation network amongst HOTAIR, miR-206 and STC2 were identified. Next, effect of HOTAIR on cell biological functions of HNSCC was identified after transfection of cells with HOTAIR overexpressed plasmids or siRNA against HOTAIR. PI3K/AKT signalling pathway-related gene expression was measured after miR-206 and STC2 were suppressed. Cell invasion, migration and proliferation were assessed. Finally, tumour growth was assessed to determine the effects of HOTAIR/miR-206/STC2 axis in vivo. RESULTS: HOTAIR specifically bound to miR-206 and miR-206 targeted STC2. Downregulated HOTAIR or upregulated miR-206 suppressed HNSCC cell proliferation, invasion and migration. miR-206 inhibited PI3K/AKT signalling pathway by down-regulating STC2. Besides, silenced HOTAIR or overexpressed miR-206 repressed the tumour growth of nude mice with HNSCC. CONCLUSION: HOTAIR regulated HNSCC cell biological functions by binding to miR-206 through STC2.


Asunto(s)
Glicoproteínas/metabolismo , Neoplasias de Cabeza y Cuello/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo
18.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-31331074

RESUMEN

In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.


Asunto(s)
Diferenciación Celular , Condrocitos/citología , Condrocitos/metabolismo , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Animales , Biomarcadores , Diferenciación Celular/genética , Células Cultivadas , Glicosilación , Hipertrofia , Metabolómica/métodos , Ratones , Osteogénesis/genética , Proteómica/métodos
19.
J Clin Pathol ; 72(10): 696-704, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31331953

RESUMEN

AIMS: Zinc-alpha 2-glycoprotein (AZGP1) is a promising tissue biomarker to predict outcomes in men undergoing treatment for localised prostate cancer (PCa). We aimed to examine the association between AZGP1 expression and the endpoints: risk of biochemical failure (BF), initiating castration-based treatment, developing castration-resistant PCa (CRPC) and PCa-specific mortality following radical prostatectomy (RP). METHODS: The study included a prospective cohort of 302 patients who underwent RP for PCa from 2002 to 2005. AZGP1 expression was analysed using immunohistochemistry on tissue microarray RP specimens and was scored semiquantitively as low or high expression. Risk of all endpoints was analysed using stratified cumulative incidences and cause-specific Cox regression, and validated with receiver operating curves, calibration and discrimination in competing-risk analyses. A meta-analysis was performed including previous studies investigating AZGP1 expression and risk of BF following RP. RESULTS: Median time of follow-up was 14.0 years. The cumulative incidence of all endpoints was significantly higher in patients with low AZGP1 expression compared with patients with high AZGP1 expression (p<0.001). In a multivariate analysis, low AZGP1 expression increases the risk of BF (HR 2.7; 95% CI 1.9 to 3.8; p<0.0001), castration-based treatment (HR 2.2; 95% CI 1.2 to 4.2; p=0.01) and CRPC (HR 2.3; 95% CI 1.1 to 5.0; p=0.03). Validation showed a low risk of prediction error and a high model performance for all endpoints. In a meta-analysis, low AZGP1 was associated with BF (HR 1.7; 95% CI 1.2 to 2.5). CONCLUSIONS: Low AZGP1 expression is associated with the risk of aggressive time-dependent outcomes in men undergoing RP for localised PCa.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Neoplasias de la Próstata/metabolismo , Estudios de Cohortes , Humanos , Inmunohistoquímica , Masculino , Análisis Multivariante , Estudios Prospectivos , Próstata/metabolismo , Próstata/patología , Prostatectomía , Neoplasias de la Próstata/cirugía , Análisis de Matrices Tisulares
20.
Nat Commun ; 10(1): 3275, 2019 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-31332201

RESUMEN

The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.


Asunto(s)
Biología Computacional/métodos , Glicómica/métodos , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Animales , Biología Computacional/normas , Bases de Datos Factuales/normas , Bases de Datos Factuales/estadística & datos numéricos , Humanos , Espectrometría de Masas/métodos , Estándares de Referencia
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