RESUMEN

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy­induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH­MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH­MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Senescencia Celular/genética , Mucoproteínas/análisis , Proteínas Oncogénicas/análisis , Biomarcadores/análisis , Biomarcadores/sangre , Neoplasias de la Mama/genética , Línea Celular Tumoral/citología , Línea Celular Tumoral/metabolismo , Senescencia Celular/fisiología , Quimioterapia/normas , Quimioterapia/estadística & datos numéricos , Femenino , Humanos , Mucoproteínas/sangre , Proteínas Oncogénicas/sangre

RESUMEN

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

Asunto(s)

Técnicas Biosensibles , Mucoproteínas/análisis , Proteínas Oncogénicas/análisis , Anticuerpos Monoclonales , Técnicas Electroquímicas , Electrodos , Oro , Humanos , Límite de Detección , Nanopartículas del Metal , Neoplasias

RESUMEN

BACKGROUND: Real life data regarding pharmacokinetics of vedolizumab in patients needing dose optimisation are scarce. We set to examine whether pre-optimisation vedolizumab levels associate with therapy outcomes and which mechanisms explain the associations. METHODS: A multicentre observational study assessed the outcome of dose increase in association with pre-escalation levels in vedolizumab-treated patients. SubsequentIy, α4ß7 occupancy on peripheral blood [PB] and intestinal lamina propria [LP] tissues was investigated on various cellular subsets in patients undergoing lower endoscopy on infusion day. Cellular localisation of vedolizumab-bound α4ß7 and effects on M1 and M2 macrophages were also explored. RESULTS: A total of 161 inflammatory bowel disease [IBD] patients were included. Among 129/161 patients intensified during maintenance [Week 14 onward], pre-intensification trough levels were comparable or higher among those subsequently attaining post-optimisation clinical, biomarker, and endoscopic remission, compared with non-remitting patients [p = 0.09, 0.25, 0.04, respectively]. Similar results were demonstrated for those dose-optimised during induction [Week 6, n = 32]. In the immune sub-study [n = 43], free α4ß7 receptors at trough were similarly low among patients with/without mucosal healing, on PB T cells [p = 0.15], LP T cells [p = 0.88], and on PB eosinophils [p = 0.08]. Integrin receptors on M1 and M2 macrophages were also saturated by low levels of vedolizumab and anti-inflammatory cytokine secretion was not increased. Co-localisation and dissociation experiments demonstrated membranal α4ß7 receptors of two origins: non-internalised and newly generated α4ß7, but re-binding was still complete at very low concentrations. CONCLUSIONS: These results do not support pharmacokinetics as the mechanism responsible for loss of response to vedolizumab, nor do they support a need for higher drug concentration to enhance vedolizumab's immune effects. Higher pre-escalation levels may indicate less clearance [less severe disease] and higher likelihood of subsequent re-gained response, regardless of therapy escalation.

Asunto(s)

Anticuerpos Monoclonales Humanizados/administración & dosificación , Fármacos Gastrointestinales/administración & dosificación , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Adulto , Biomarcadores/análisis , Proteína C-Reactiva/análisis , Moléculas de Adhesión Celular/análisis , Relación Dosis-Respuesta a Droga , Endoscopía Gastrointestinal , Femenino , Humanos , Macrófagos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mucoproteínas/análisis , Albúmina Sérica/análisis

RESUMEN

BACKGROUND AND AIMS: Some patients with ulcerative colitis [UC] do not respond to vedolizumab treatment despite adequate drug exposure in serum. This study aimed to investigate vedolizumab in tissue and questioned whether insufficient tissue exposure could explain non-response in UC patients with adequate serum vedolizumab concentrations. METHODS: A paired serum sample and colonic mucosal biopsy was collected from 40 UC patients [20 endoscopic responders, 20 non-responders] at week 14 of vedolizumab treatment. Vedolizumab, soluble [s]-mucosal addressin cell adhesion molecule-1 [MAdCAM-1], s-vascular cell adhesion molecule-1 [VCAM-1] and s-intercellular adhesion molecule-1 [ICAM-1] were measured in serum and/or tissue. Endoscopic response was defined as Mayo endoscopic sub-score ≤1. RESULTS: A significant positive correlation was observed between vedolizumab serum and colonic tissue concentrations [ρ = 0.84, p < 0.0001], regardless of the macroscopic inflammatory state of the tissue. Vedolizumab tissue concentrations were lower in non-responders than in responders [0.07 vs 0.11 µg/mg, p = 0.04]. In the subgroup of patients with adequate vedolizumab serum concentrations [>14.6 µg/mL], tissue vedolizumab was not significantly different between responders and non-responders [0.15 vs 0.13 µg/mg; p = 0.92]. Serum sMAdCAM-1 concentrations, but not serum sICAM-1 or sVCAM-1 concentrations, were significantly higher in responders than in non-responders with adequate vedolizumab serum concentrations [1.04 vs 0.83 ng/mL, p = 0.03]. CONCLUSIONS: Vedolizumab concentrations in colonic mucosal tissue of UC patients reflect the concentration in serum regardless of the macroscopic inflammatory state of the tissue. Our data show that insufficient tissue exposure does not explain non-response in UC patients with adequate serum vedolizumab concentrations.

Asunto(s)

Anticuerpos Monoclonales Humanizados , Moléculas de Adhesión Celular , Colitis Ulcerosa , Colon/patología , Endoscopía Gastrointestinal , Mucosa Intestinal , Mucoproteínas , Adulto , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/farmacocinética , Biopsia/métodos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/metabolismo , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Monitoreo de Drogas/métodos , Endoscopía Gastrointestinal/métodos , Endoscopía Gastrointestinal/estadística & datos numéricos , Femenino , Fármacos Gastrointestinales/administración & dosificación , Fármacos Gastrointestinales/farmacocinética , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Mucoproteínas/análisis , Mucoproteínas/metabolismo , Inducción de Remisión , Distribución Tisular , Insuficiencia del Tratamiento

RESUMEN

Arabinogalactan proteins (AGPs) are important plant proteoglycans involved in many development processes. In roots, AGPs occur in the cell wall of root cells and root cap-derived cells as well as in the secreted mucilage. Detection, localization , and quantification techniques are therefore essential to unravel the AGP diversity of structures and functions. This chapter details root-adapted immunocytochemical methods using monoclonal antibodies, and a collection of biochemical analysis protocols using ß-D-glucosyl Yariv reagent for comprehensive AGP characterization.

Asunto(s)

Cromatografía en Gel/métodos , Electroforesis/métodos , Inmunohistoquímica/métodos , Mucoproteínas/análisis , Arabidopsis/química , Pared Celular/química , Glucósidos/química , Mucoproteínas/química , Pisum sativum/química , Floroglucinol/análogos & derivados , Floroglucinol/química , Mucílago de Planta/análisis , Mucílago de Planta/química , Proteínas de Plantas/análisis , Proteínas de Plantas/química , Raíces de Plantas/química

RESUMEN

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

Asunto(s)

Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mucoproteínas/análisis , Mucoproteínas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , ADN/análisis , ADN/aislamiento & purificación , Indoles/química , Mucoproteínas/metabolismo , Óvulo Vegetal/citología , Óvulo Vegetal/genética , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN/análisis , ARN/metabolismo , Sondas ARN/síntesis química , Sondas ARN/metabolismo

RESUMEN

Arabinogalactan proteins are a diverse group of cell wall-associated proteoglycans. While structural and molecular genetic analyses have contributed to the emerging improved understanding of the wide-range of biological processes in which AGPs are implicated; the ability to detect, localize, and quantify them is fundamentally important. This chapter describes three methods: histological staining, radial gel diffusion, and colorimetric quantification, each of which utilize the ability of Yariv reagent to bind to AGPs.

Asunto(s)

Proteínas Algáceas/química , Mucoproteínas/análisis , Mucoproteínas/aislamiento & purificación , Proteínas Algáceas/análisis , Proteínas Algáceas/aislamiento & purificación , Proteínas Algáceas/metabolismo , Pared Celular/química , Colorimetría/métodos , Glucósidos , Inmunodifusión/métodos , Mucoproteínas/metabolismo , Floroglucinol/análogos & derivados , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Coloración y Etiquetado/métodos

RESUMEN

Winemaking results in a significant amount of sediments that are formed in the tanks, the vats and in the bottles before and after fermentation. Little is known about the biochemical composition of these sediments apart from the fact that they are assumed to be derived in large part from the grape matrix. Glycan microarray technology offers a relatively rapid means to track the polysaccharides from their origin in the grape material and throughout the various steps in the winemaking process. In this study Comprehensive Microarray Polymer Profiling (CoMPP) was used to investigate the glycan-rich composition of particularly white grapes during winemaking and then investigate the effects of recombinant and commercial enzyme formulations on wine sediment compositions. The gross lees or sediments produced in the absence of enzymes were found to be composed of an abundance of homogalacturonans, rhamnogalacturonans, arabinans and galactans in addition to an abundance of extensins and arabinogalactan proteins. The addition of enzymes was shown to strip off the homogalacturonan and much of the rhamnogalacturonan with its side chains revealing a sediment layer composed almost exclusively of extensins and arabinogalactan proteins. The effect of winemaking techniques was shown to have an effect on the glycan-rich wine sediment compositions and holds implications for the management of gross lees in a winery environment.

Asunto(s)

Fermentación , Glicoproteínas/análisis , Polisacáridos/química , Vitis/química , Vino/análisis , Pared Celular/química , Enzimas/metabolismo , Frutas/química , Galactanos , Mucoproteínas/análisis , Proteínas de Plantas/análisis

RESUMEN

AIM: the number of intestinal IgA+ lymphocytes are decreased in acute liver necrosis and the mechanism remains poorly understood. The purpose of this study was to observe the role of lymphocyte homing and apoptosis associated with decreased intestinal IgA positive lymphocytes in acute liver necrosis. METHODS: the acute liver necrosis mouse model and LTßR pre-treatment were used to assess intestinal mucosal addressin cell adhesion molecule-1 (MAdCAM - 1) expression, cell apoptosis, IgA+ cells and secretory immunoglobulin A (SIgA). RESULTS: MAdCAM - 1 mRNA and protein expression decreased significantly in the acute necrosis group; 0.57 ± 0.032 fold vs. baseline (p < 0.05) and 0.45 ± 0.072 fold vs. baseline (p < 0.05), respectively. LTßR pre-treatment could significantly improve the decline of MAdCAM - 1 mRNA and protein expression in the intestinal mucosa (1.83 ± 0.064 fold vs. baseline, p < 0.05 and 1.75 ± 0.046 fold vs. baseline, p < 0.05, respectively) and partially restore the decline in IgA+ lymphocytes and SIgA levels. There were increased rates of enterocyte apoptosis in both the acute liver necrosis and LTßR pre-treatment group; 0.79% vs. control (p < 0.05) and 0.77% vs. control (p < 0.05), respectively). CONCLUSION: our results suggest that the dysfunction of lymphocyte homing and apoptosis are both involved with decreased intestinal IgA+ lymphocytes in acute liver necrosis. LTßR pre-treatment can partially restore IgA+ cells and SIgA by increasing MAdCAM - 1 expression, rather than inhibiting lymphocyte apoptosis.

Asunto(s)

Moléculas de Adhesión Celular/análisis , Movimiento Celular/fisiología , Inmunoglobulina A/análisis , Hígado/patología , Linfocitos/fisiología , Mucoproteínas/análisis , Alanina Transaminasa/sangre , Animales , Apoptosis , Aspartato Aminotransferasas/sangre , Moléculas de Adhesión Celular/genética , Movimiento Celular/inmunología , Modelos Animales de Enfermedad , Enterocitos/fisiología , Inmunidad Mucosa/inmunología , Inmunoglobulina A Secretora/análisis , Etiquetado Corte-Fin in Situ , Hígado/inmunología , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mucoproteínas/genética , Necrosis/inmunología , Necrosis/patología , ARN Mensajero/análisis

RESUMEN

MAIN CONCLUSION: RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

Asunto(s)

Mucoproteínas/análisis , Pectinas/análisis , Polisacáridos/análisis , Populus/metabolismo , Pared Celular/metabolismo , Mananos/análisis , Mananos/metabolismo , Mucoproteínas/metabolismo , Pectinas/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismo , Populus/citología , Populus/crecimiento & desarrollo , Árboles , Madera/citología , Madera/genética , Madera/metabolismo , Xilema/citología , Xilema/crecimiento & desarrollo , Xilema/metabolismo

RESUMEN

Arabinogalactan proteins (AGP) and pectic polysaccharides were isolated from above-ground parts of Heracleum sosnowskyi. The structural study has shown that a linear region of the pectic macromolecules consists of 1,4-α-d-galactopyranosyluronan blocks partially methyl esterified and acetylated. The branched region consists of 3-O- and partially 2-O-acetylated rhamnogalacturonan I. Side chains of the RG-I backbone include the regions of arabinogalactan I and branched 1,5-α-l-arabinan. The carbohydrate part of AGP consists of arabinogalactan II with a 1,3-ß-d-Galp main chain. The side chains of the branched area of AG-II are composed of 1,6-ß-d-Galp, 1,5-, 1,3,5-α-l-Araf, 4-O-Me-ß-d-GlcA and 1,4-ß-d-GlcpA, and non-reducing ends residues of ß-d-Galp, α-l-Araf, α-l-Rhap and α-l-Fucp. The branch points of the main and side chains are formed by 3,6-di-O-substituted ß-d-Galp. It was found that at least a portion of pectin is probably covalently linked to AGP, wherein AGP is linked to RG-I, but not with galacturonan.

Asunto(s)

Heracleum/química , Mucoproteínas/análisis , Proteínas de Plantas/análisis , Polisacáridos/análisis , Galactanos/análisis , Espectroscopía de Resonancia Magnética , Oxalatos/química , Pectinas/análisis , Solubilidad

RESUMEN

MAIN CONCLUSION: Both male and female gametes of archegoniates are highly specialized cells surrounded by an extraprotoplasmic matrix rich in AGPs, which are speculated to facilitate development and gamete fusion through Ca 2+) oscillations. An additional layer, the egg envelope, forms around the egg periphery, except at the fertilization pore, and contains arabinose-rich polymers that presumably impart flexibility for the rapidly growing zygote and embryo. The abundant AGPs and arabinan pectins associated with the eggs of C. richardii not only are integral to development, fertilization, and early embryogenesis, but also may be involved in desiccation tolerance important to the survival of the reproductive gametophyte. A defining feature of gametogenesis in archegoniates is the deposition of a special matrix outside of the plasmalemma of both egg and sperm cells that displaces the primary cell wall away from the protoplasm. It is within this matrix that gamete differentiation occurs. In leptosporangiate ferns, maturation of the egg cell involves the deposition of a second specialized wall, the so-called egg envelope that surrounds the cell except at the fertilization pore, a narrow site where gamete fusion takes place. We provide the first conclusive evidence of the macromolecular constituents in the unique structures surrounding fern egg cells before and after fertilization. To test the hypotheses that the egg extracellular matrix contains arabinogalactan proteins (AGPs) as does the sperm cell matrix, and that cell wall polysaccharides, especially pectins, are components of the egg envelope, we examined the expression patterns of AGPs and cell wall constituents during oogenesis in Ceratopteris richardii. Utilizing histochemical stains for callose, cellulose and AGPs coupled with immunogold localizations employing a suite of monoclonal antibodies to cell wall components (JIM13, JIM8, LM2, LM5, LM6, LM19, LM20 and anticallose), we demonstrate that AGPs, but not pectins, are abundant in the matrix around egg cells and degrading neck canal and ventral canal cells during archegonial development. A striking finding is that both AGPs and (1,5)-α-L-arabinan pectin epitopes are principle components of the egg envelope before and after fertilization, suggesting that they are important in both egg maturation and gamete fusion.

Asunto(s)

Mucoproteínas/análisis , Óvulo Vegetal/química , Pectinas/metabolismo , Pteridaceae/química , Anticuerpos Monoclonales/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Epítopos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glucanos/metabolismo , Microscopía Electrónica de Transmisión , Mucoproteínas/inmunología , Mucoproteínas/metabolismo , Óvulo Vegetal/metabolismo , Pectinas/análisis , Pectinas/inmunología , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Polisacáridos/análisis , Polisacáridos/metabolismo , Pteridaceae/metabolismo

RESUMEN

Nodular gastritis is a form of chronic Helicobacter pylori gastritis affecting the gastric antrum and characterised endoscopically by the presence of small nodular lesions resembling gooseflesh. It is generally accepted that hyperplasia of lymphoid follicles histologically characterises nodular gastritis; however, quantitative analysis in support of this hypothesis has not been reported. Our goal was to determine whether nodular gastritis is characterised by lymphoid follicle hyperplasia.The number, size, and location of lymphoid follicles in nodular gastritis were determined and those properties compared to samples of atrophic gastritis. The percentages of high endothelial venule (HEV)-like vessels were also evaluated.The number of lymphoid follicles was comparable between nodular and atrophic gastritis; however, follicle size in nodular gastritis was significantly greater than that seen in atrophic gastritis. Moreover, lymphoid follicles in nodular gastritis were positioned more superficially than were those in atrophic gastritis. The percentage of MECA-79 HEV-like vessels was greater in areas with gooseflesh-like lesions in nodular versus atrophic gastritis.Superficially located hyperplastic lymphoid follicles characterise nodular gastritis, and these follicles correspond to gooseflesh-like nodular lesions observed endoscopically. These observations suggest that MECA-79 HEV-like vessels could play at least a partial role in the pathogenesis of nodular gastritis.

Asunto(s)

Mucosa Gástrica/patología , Gastritis/patología , Tejido Linfoide/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Superficie/análisis , Antígenos de Superficie/biosíntesis , Moléculas de Adhesión Celular , Niño , Preescolar , Endotelio Vascular/metabolismo , Femenino , Gastritis/microbiología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori , Humanos , Inmunoglobulinas/análisis , Inmunoglobulinas/biosíntesis , Inmunohistoquímica , Tejido Linfoide/irrigación sanguínea , Tejido Linfoide/metabolismo , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/biosíntesis , Persona de Mediana Edad , Mucoproteínas/análisis , Mucoproteínas/biosíntesis , Adulto Joven

RESUMEN

Here we determined whether immunostimulatory plant-derived arabinogalactan proteins (AGPs) and the honeybee-derived protein apisimin are present in varieties of New Zealand honey. Apisimin is a protein of unknown function secreted from the glands of honeybees into Royal Jelly, forming a complex with apalbumin1 capable of stimulating lymphocyte proliferation. AGPs were abundant in kanuka honey with lesser amounts in manuka, kowhai and clover honeys, but absent from Royal Jelly. Apisimin was present in all honeys, as well as Royal Jelly. We report that apisimin shares with honey AGPs the ability to stimulate the release of TNF-α from blood monocytes. Further, it synergizes with AGPs to enhance the release of TNF-α, via a mechanism not involving the formation of a complex with AGPs. In summary, this study provides evidence that AGPs and apisimin are commonly present in different floral varieties of honey, and hence contribute to their immunostimulatory properties.

Asunto(s)

Miel/análisis , Proteínas de Insectos/análisis , Monocitos/inmunología , Mucoproteínas/análisis , Animales , Abejas , Células Cultivadas , Ácidos Grasos/análisis , Ácidos Grasos/inmunología , Humanos , Proteínas de Insectos/inmunología , Proteínas de Insectos/farmacología , Monocitos/efectos de los fármacos , Mucoproteínas/inmunología , Nueva Zelanda , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

Asunto(s)

Pared Celular/metabolismo , Glicoproteínas/genética , Mucoproteínas/genética , Nicotiana/citología , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Secuencia de Bases , Pared Celular/química , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Glicosilación , Datos de Secuencia Molecular , Mucoproteínas/análisis , Mucoproteínas/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/química , Nicotiana/metabolismo , Regulación hacia Arriba

RESUMEN

Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobial activity, produced predominantly by lactic acid bacteria, with potential applications as biopreservatives and probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach using nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentation techniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillus acidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collision dissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusion sample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or related proteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin D were predominant. The process of freeze drying under vacuum was observed to affect both the diversity and abundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentation modes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverage and number of bacteriocin peptides identified. Multi-enzyme proteolytic digestion was observed to increase the sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MS analysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic data analysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.

Asunto(s)

Bacteriocinas/análisis , Lactobacillus acidophilus , Probióticos/química , Cromatografía Liquida , Biología Computacional , Digestión , Estudios de Evaluación como Asunto , Liofilización , Peso Molecular , Mucoproteínas/análisis , Péptido Hidrolasas/metabolismo , Proteolisis , Proteómica , Espectrometría de Masas en Tándem

RESUMEN

BACKGROUND: Because dacryoliths occur at low frequency, few studies have focused on their composition. We aimed to present findings from morphological, chemical, and mineralogic analysis of 86 dacryoliths. METHODS: We studied 86 dacryoliths obtained during 832 dacryocystorhinostomies (DCR) performed for postsaccal obstruction. We examined the samples with atomic infrared spectrometry (80 samples), amino acid analysis (17 samples), scanning electron microscopy, and an electron microprobe with an energy dispersive detector (seven samples). RESULTS: Dacryoliths were found in 86/832 DCRs (10.3 %), mostly in patients with primary acquired nasolacrimal duct obstruction. All the dacryoliths were soft, composed of organic material, including proteins and mucoproteins, with approximately 20 % amino acid content. There were no "hard" dacryoliths composed of calcium phosphate. The stones were composed of lobes and lobules built on an amorphous core material with small cavities, probably as a result of various chemical processes that produced a gaseous product. The most frequent elements found in inorganic inclusions were silicon, magnesium, sulfur, potassium, calcium, sodium, and chlorine. Also, some particles had high contents of bismuth, titanium, iron, and organic fibers. The fibers found in the core of dacryoliths suggested a potential origin from cotton swabs used in cosmetics. CONCLUSION: Dacryoliths are composed almost exclusively of organic material, including proteins and mucoproteins, with approximately 20 % amino acid content.

Asunto(s)

Cálculos/química , Enfermedades del Aparato Lagrimal/diagnóstico , Litiasis/química , Litiasis/diagnóstico , Conducto Nasolagrimal/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Aminoácidos/análisis , Calcio/análisis , Cálculos/ultraestructura , Niño , Preescolar , Dacriocistorrinostomía , Microanálisis por Sonda Electrónica , Femenino , Humanos , Lactante , Enfermedades del Aparato Lagrimal/cirugía , Litiasis/cirugía , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Mucoproteínas/análisis , Espectroscopía Infrarroja por Transformada de Fourier , Adulto Joven

RESUMEN

Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18. We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years.

Asunto(s)

Gastrópodos/química , Espectrometría de Masas/métodos , Mucoproteínas/análisis , Análisis de Secuencia de ADN/métodos , Secuencia de Aminoácidos , Animales , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Datos de Secuencia Molecular , Mucoproteínas/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Mapeo Peptídico , ARN/análisis , Alineación de Secuencia , Espermatozoides/química , Testículo/química , Transcriptoma

RESUMEN

The present study aimed at analyzing the structural features of seed mucilage and cell-wall polysaccharides which accounted for 41% of the mass of flax meal (FM). A combination of high molar-mass mucilage-like polysaccharides (rhamnogalacturonan and arabinoxylan) was released from FM in water, together with arabinogalactan proteins and glucans. About half of FM homogalacturonans was extracted using a calcium chelator and boiling water. Hemicellulosic xyloglucans and xylans were further extracted with 1M KOH, in ∼13% FM-sugars yield. Structural characterization of the xyloglucan using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy showed the presence of XXXG type xyloglucan, but also that of XXGG-structure, possibly characteristic of flax seeds. Hydrolysis of xylans with endo-(1→4)-ß-D-xylanase, and analysis of the neutral and acidic oligosaccharides by MALDI-TOF-MS showed that xylan consisted of ß-(1→4)-linked-D-xylopyranose backbone with some zones (DP 5-7) substituted with 4-O-MeGlcA\GlcA\Glc residues.

Asunto(s)

Pared Celular/química , Lino/química , Mucoproteínas/análisis , Mucílago de Planta/análisis , Polisacáridos/análisis , Semillas/química , Quelantes/química , Hidrólisis , Mucoproteínas/química , Pectinas/química , Mucílago de Planta/química , Proteínas de Plantas/análisis , Proteínas de Plantas/química , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Ácidos Urónicos/química , Agua/química , Xilanos/química

RESUMEN

OBJECTIVES: Type 1 autoimmune pancreatitis (AIP) is histologically characterized by dense lymphoplasmacytic infiltration and marked storiform fibrosis, manifestations associated with pancreatic ducts. Such periductal lymphocyte recruitment is thought to be elicited by dysregulation of mechanisms governing physiological lymphocyte homing. The present study was undertaken to determine whether vascular addressins including peripheral lymph node addressin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) play a role in type 1 AIP histogenesis. METHODS: Tissue sections of type 1 AIP and tumor-associated non-AIP chronic pancreatitis, as well as normal pancreas, were subjected to immunohistochemical analysis using vascular addressin-related antibodies. RESULTS: The number of periductal mouse endothelial cell antigen 79-positive high endothelial venule (HEV)-like vessels was increased in type 1 AIP relative to that seen in non-AIP chronic pancreatitis, whereas the number of MAdCAM-1-positive HEV-like vessels did not differ between the 2 conditions. Mouse endothelial cell antigen 79 antigens are expressed on duct-forming epithelial cells not only in pancreas but also in salivary glands, which often harbor extrapancreatic lesions in type 1 AIP. CONCLUSIONS: Type 1 AIP can be characterized by periductal induction of MECA-79-positive HEV-like vessels. MECA-79-positive 6-sulfo sialyl Lewis X-related carbohydrate antigens expressed on duct-forming epithelial cells could be associated with type 1 AIP pathogenesis.

Asunto(s)

Enfermedades Autoinmunes/patología , Vasos Linfáticos/patología , Conductos Pancreáticos/patología , Pancreatitis/patología , Antígenos CD34/análisis , Antígenos de Superficie/análisis , Enfermedades Autoinmunes/metabolismo , Biomarcadores/análisis , Moléculas de Adhesión Celular , Humanos , Inmunoglobulina G/análisis , Inmunoglobulinas/análisis , Inmunohistoquímica , Queratinas/análisis , Vasos Linfáticos/química , Proteínas de la Membrana/análisis , Mucoproteínas/análisis , Conductos Pancreáticos/química , Pancreatitis/metabolismo