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AIM: We investigated the expression of neuropeptide Y (NPY), NPY receptor 1 (NPYR1) and NPY receptor 2 (NPYR2) in infantile haemangiomas (IHs). METHODS: Immunohistochemical (IHC) staining was performed on proliferating IHs from six patients aged 4-13 (mean 8.7) months and involuted IHs from six patients aged 5-59 (mean 18.7) years, for the expression of NPY, NPYR1 and NPYR2. Protein and messenger ribonucleic acid expression corresponding to these proteins was investigated by Western blotting and NanoString analysis, respectively. RESULTS: IHC staining, Western blotting and NanoString analysis demonstrated the presence of NPYR1, but not NPYR2, within proliferating and involuted IHs. IHC staining showed NPYR1 was expressed by B and T lymphocytes expressing CD45 and mast cells expressing tryptase. IHC staining demonstrated the presence of NPY on the NPYR1+ cells, but it was not detected by Western blotting or NanoString analysis. CONCLUSION: NPYR1, but not NPYR2, was present in IHs. The localisation of NPYR1 to B and T lymphocytes and mast cells suggests its role in the biology of IHs. The demonstration of NPY on the NPYR1+ cells, without active transcription, suggests that NPY was not being produced within IHs.
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Linfócitos B/metabolismo , Hemangioma/imunologia , Mastócitos/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Linfócitos T/metabolismo , Adolescente , Adulto , Western Blotting , Contagem de Células , Criança , Pré-Escolar , Expressão Gênica , Hemangioma/metabolismo , Hemangioma/patologia , Humanos , Imuno-Histoquímica , Adulto JovemRESUMO
BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in men and the third leading cause of cancer related deaths among men living in developed countries. Biomarkers that predict disease outcome at the time of initial diagnosis would substantially aid disease management. RESULTS: Proteins extracted from formalin-fixed paraffin-embedded tissue were identified using nanoflow liquid chromatography-MALDI MS/MS or after separation by one- or two-dimensional electrophoresis. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000963. A list of potential biomarker candidates, based on proposed associations with prostate cancer, was derived from the 320 identified proteins. Candidate biomarkers were then examined by multiplexed Western blotting of archival specimens from men with premetastatic disease and subsequent disease outcome data. Annexin A2 provided the best prediction of risk of metastatic disease (log-rank Chi squared p = 0. 025). A tumor/control tissue >2-fold relative abundance increase predicted early biochemical failure, while <2-fold change predicted late or no biochemical failure. CONCLUSIONS: This study confirms the potential for use of archival FFPE specimens in the search for prognostic biomarkers for prostate cancer and suggests that annexin A2 abundance in diagnostic biopsies is predictive for metastatic potential. Protein profiling each cancer may lead to an overall reduction in mortality from metastatic prostate cancer as well as reduced treatment associated morbidity.
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Plant polysaccharide-degrading rumen microbes are fundamental to the health and productivity of ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-producing anaerobic bacterium with a key role in hemicellulose degradation in the rumen. Gel-based proteomics was used to examine the growth-phase-dependent abundance patterns of secreted proteins recovered from cells grown in vitro with xylan or xylose provided as the sole supplementary carbon source. Five polysaccharidases and two carbohydrate-binding proteins (CBPs) were among 30 identified secreted proteins. The endo-1,4-ß-xylanase Xyn10B was 17.5-fold more abundant in the culture medium of xylan-grown cells, which suggests it plays an important role in hemicellulose degradation. The secretion of three nonxylanolytic enzymes and two CBPs implies they augment hemicellulose degradation by hydrolysis or disruption of associated structural polysaccharides. Sixteen ATP-binding cassette (ABC) transporter substrate-binding proteins were identified, several of which had altered relative abundance levels between growth conditions, which suggests they are important for oligosaccharide uptake. This study demonstrates that B. proteoclasticus modulates the secretion of hemicellulose-degrading enzymes and ATP-dependent sugar uptake systems in response to growth substrate and supports the notion that this organism makes an important contribution to polysaccharide degradation in the rumen.
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Proteínas de Bactérias/metabolismo , Butyrivibrio/enzimologia , Glicosídeo Hidrolases/metabolismo , Proteoma/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Meios de Cultivo Condicionados/química , Glicosídeo Hidrolases/química , Lignina , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Proteólise , Proteoma/química , Rúmen/microbiologia , Xilanos/químicaRESUMO
AIM: We investigated the expression of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations we have identified in moderately differentiated lip squamous cell carcinoma (MDLSCC). METHOD: Ten MDLSCC samples underwent 3,3-diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and receptor 2 (ATIIR2). NanoString analysis and Western blotting (WB) were performed on six MDLSCC samples for gene and protein expression, respectively. RESULTS: IHC staining showed expression of PRR, ATIIR1, and ATIIR2 on cells within the tumor nests (TNs) and the stroma. ACE was localized to the microvessels within the stroma. WB detected PRR, ACE, and ATIIR2. NanoString analysis confirmed gene expression of PRR, ACE, and ATIIR1. CONCLUSION: Components of the RAS: PRR, ATIIR1, and ATIIR2 are expressed on two CSC subpopulations in MDLSCC, one within the TNs and the other within the stroma. The endothelium of the microvessels within the stroma expresses ACE.
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AIM: To identify and characterize cancer stem cells (CSCs) in moderately differentiated lip squamous cell carcinoma (MDLSCC). METHOD: MDLSCC samples underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for squamous cell carcinoma marker EMA, CSC marker CD44 and embryonic stem cell markers NANOG, octamer-binding transcription factor 4 (OCT4), spalt-like transcription factor 4 (SALL4), sex-determining region Y-box 2 (SOX2), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Immunofluorescent IHC staining was performed on two MDLSCC samples. Western blotting (WB) was used to confirm the expression of the aforementioned proteins and their transcription activation was investigated using NanoString and RT-qPCR. RESULTS: IHC staining demonstrated the presence of (1) an EMA+/CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4- CSC subpopulation within the tumor nests (TNs); (2) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4- CSC subpopulation; and (3) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4+ CSC subpopulation within the stroma, between the TNs. NanoString and RT-qPCR confirmed the presence of mRNA for CD44, SALL4, STAT3, SOX2, and OCT4, and WB confirmed the presence of NANOG, pSTAT3, SOX2, and OCT4. CONCLUSION: This study demonstrates three putative CSC subpopulations within MDLSCC.
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The renin-angiotensin system (RAS) mediates cardiac and renal fibrosis. Dupuytren's disease (DD) is a proliferative fibromatosis affecting the hands. This study investigated the expression of the RAS in DD. METHODS: 3,3-Diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) was performed on 4-µm thick formalin-fixed paraffin-embedded sections of DD cords and nodules from 6 patients. Western blotting (WB) and NanoString mRNA analysis were performed to confirm RAS protein expression and transcriptional activation, respectively. RESULTS: IHC staining demonstrated the expression of PRR, ACE, ATIIR1, and ATIIR2 on the ERG+ and CD34+ endothelium of the micro vessels surrounding the DD cords and nodules. PRR was also expressed on the pericyte layer of these microvessels. WB confirmed protein expression of PRR, ACE, and ATIIR2 but not ATIIR1. NanoString analysis confirmed transcriptional activation of PRR, ACE, ATIIR1, but ATIIR2 was below detectable levels. CONCLUSIONS: We demonstrated expression of PRR, ATIIR1, ATIIR2, and ACE on the embryonic stem cell-like cell population on the microvessels surrounding DD nodules and cords by IHC staining, although the expression of ATIIR1 was not confirmed by WB and that of ATIIR2 was below detectable levels on NanoString analysis. These findings suggest the embryonic stem cell-like cell population as a potential therapeutic target for DD, by using RAS modulators.
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BACKGROUND/AIM: We previously reported the use of mass spectrometry and western blotting to identify proteins from tumour regions of formalin-fixed paraffin-embedded biopsies from 16 men who presented with apparently localized prostate cancer, and found that annexin A2 (ANXA2) appeared to be a better predictor of subsequent biochemical failure than prostate-specific antigen (PSA). MATERIALS AND METHODS: In this follow-up study, ANXA2 and PSA were measured using western blotting of proteins extracted from biopsies from 37 men from a subsequent prostate cancer trial. RESULTS: No significant differences in ANXA2 and PSA levels were observed between men with and without biochemical failure. The statistical effect sizes were small, d=0.116 for ANXA2, and 0.266 for PSA. CONCLUSION: ANXA2 and PSA proteins measured from biopsy tumour regions are unlikely to be good biomarkers for prediction of the clinical outcome of prostate cancer presenting with apparently localized disease.
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Anexina A2/metabolismo , Antígeno Prostático Específico/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Biópsia , Western Blotting , Quimiorradioterapia , Humanos , Masculino , Avaliação de Resultados em Cuidados de Saúde , Prognóstico , Próstata/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapiaRESUMO
AIMS: The embryonic-like stem cell origin of infantile hemangioma (IH) and the observed elevated serum levels of alpha-fetoprotein (AFP) in patients with hepatic IH led us to investigate if this tumor was the source of AFP. MATERIALS AND METHODS: We measured serial serum levels of AFP in patients with problematic proliferating IH treated with surgical excision or propranolol treatment. We also investigated the expression of AFP in extrahepatic IH samples using immunohistochemical staining, mass spectrometry, NanoString gene expression analysis, and in situ hybridization. RESULTS: Serum levels of AFP normalized following surgical excision or propranolol treatment. Multiple regression analysis for curve fittings revealed a different curve compared to reported normal values in the general populations. AFP was not detected in any of the IH samples examined at either the transcriptional or translational levels. CONCLUSION: This study demonstrates the association of proliferating IH with elevated serum levels of AFP, which normalized following surgical excision or propranolol treatment. We have shown that IH is not the direct source of AFP. An interaction between the primitive mesoderm-derived IH and the endogenous endodermal tissues, such as the liver, via an intermediary, may explain the elevated serum levels of AFP in infants with extrahepatic IH.
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AIM: We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin-angiotensin system (RAS). METHODS: 3,3'-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC. RESULTS: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively. CONCLUSION: Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.
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AIMS: The role of the renin-angiotensin system (RAS) in the biology of infantile hemangioma (IH) represents an emerging paradigm, particularly the involvement of renin, angiotensin converting enzyme, and angiotensin II. This study investigated the expression of cathepsins B, D, and G, enzymes that may modulate the RAS, in IH. MATERIALS AND METHODS: The expression of cathepsins B, D, and G was examined using immunohistochemistry, enzyme activity assays, mass spectrometry, and NanoString gene expression assay in IH samples at different phases of development. RESULTS: Immunohistochemical staining showed the expression of cathepsins B, D, and G in proliferating and involuted IH samples. This was confirmed at the transcriptional level using NanoString gene expression assays. Mass spectrometry confirmed the identification of cathepsins D and G in all three phases of IH development, whereas cathepsin B was detected in 2/2 proliferating and 1/2 involuting lesions. Enzyme activity assays demonstrated the activity of cathepsins B and D, but not G, in all phases of IH development. CONCLUSION: Our data demonstrated the presence of cathepsins B, D, and G in IH. Their role in modulating the RAS and the biology of IH offers potential novel targets for the management of this tumor.
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AIMS: Interstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and the expression of Thy-1, a marker of thymocyte progenitors, which have the ability to give rise to both B and T cells. METHODS: Immunohistochemical (IHC) staining was performed on proliferating and involuted IHs for the expression of CD45, CD3, CD20, CD79a, Thy-1 and CD34. The presence of mRNA corresponding to CD45, CD3G, CD20 and Thy-1 was confirmed by reverse transcriptase-polymerase chain reaction in snap-frozen IH tissues. Cell counting of 3,3-diaminobenzidine IHC-stained slides was performed on CD45+ only cells and dually stained CD45+/CD3+ cells or CD45+/CD20+ cells and analysed statistically. In situ hybridisation and mass spectrometry were also performed to confirm the presence and abundance of Thy-1, respectively. RESULTS: IHC staining showed a subpopulation of CD45+ interstitial cells that expressed the T lymphocyte marker, CD3, and another subpopulation that expressed the B lymphocyte marker, CD20, in proliferating and diminished in involuted IHs. The abundant expression of Thy-1 on the endothelium of proliferating, but not involuted IH, was demonstrated by IHC staining and confirmed by in situ hybridisation and mass spectrometry. CONCLUSIONS: Both B and T lymphocytes are present within the interstitium of proliferating and involuted IH. The expression of Thy-1 by the endothelium suggests that B and T cells in IH may have originated from within the lesion, rather than migrating from the peripheral circulation.
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Subpopulações de Linfócitos B/imunologia , Biomarcadores Tumorais/análise , Hemangioma/imunologia , Subpopulações de Linfócitos T/imunologia , Antígenos CD20/análise , Subpopulações de Linfócitos B/patologia , Biomarcadores Tumorais/genética , Complexo CD3/análise , Linhagem da Célula , Proliferação de Células , Células Endoteliais/imunologia , Células Endoteliais/patologia , Hemangioma/genética , Hemangioma/patologia , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização In Situ , Lactente , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/genética , Fenótipo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/patologia , Espectrometria de Massas em Tandem , Antígenos Thy-1/análise , Antígenos Thy-1/genéticaRESUMO
The growth and productivity of ruminants depends on a complex microbial community found in their fore-stomach (rumen), which is able to breakdown plant polysaccharides and ferment the released sugars. Butyrivibrio proteoclasticus B316T is a Gram-positive polysaccharide-degrading, butyrate-producing bacterium that is present at high numbers in the rumen of animals consuming pasture or grass silage based diets. B316T is one of a small number of rumen fibrolytic microbes capable of efficiently degrading and utilizing xylan, as well as being capable of utilizing arabinose, xylose, pectin and starch. We have therefore carried out a proteomic analysis of B316T to identify intracellular enzymes that are implicated in the metabolism of internalized xylan. Three hundred and ninety four proteins were identified including enzymes that have potential to metabolize assimilated products of extracellular xylan digestion. Identified enzymes included arabinosidases, esterases, an endoxylanase, and ß-xylosidase. The presence of intracellular debranching enzymes indicated that some hemicellulosic side-chains may not be removed until oligosaccharides liberated by extracellular digestion have been assimilated by the cells. The results support a model of extracellular digestion of hemicellulose to oligosaccharides that are then transported to the cytoplasm for further digestion by intracellular enzymes.
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The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos/fisiologia , Clostridium/metabolismo , Animais , Transporte Biológico/fisiologia , Rúmen/microbiologia , Ruminantes/microbiologiaAssuntos
Carcinoma de Células Escamosas/metabolismo , Células-Tronco Neoplásicas/patologia , Sistema Renina-Angiotensina , Neoplasias da Língua/metabolismo , Língua/patologia , Carcinoma de Células Escamosas/diagnóstico , Diferenciação Celular , Humanos , Imuno-Histoquímica , Neoplasias da Língua/diagnósticoRESUMO
Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4 Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.
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Adaptação Fisiológica , Butyrivibrio/genética , Polissacarídeos/metabolismo , Adaptação Fisiológica/genética , Animais , Aderência Bacteriana/genética , Butyrivibrio/metabolismo , Genoma Bacteriano/genética , Genômica , RúmenRESUMO
One of the most significant morphogenic events in the development of Drosophila melanogaster is the elongation of imaginal discs during puparium formation. We have shown that this macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. We have shown that the fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks (V. Kondylis, S. E. Goulding, J. C. Dunne, and C. Rabouille, 2001, Mol. Biol. Cell, 12, 2308). Using mRNA in situ hybridisation, we show here that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, dsed5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). We show that the transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. And we show that the ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing our understanding of the relationship between gene expression and organelle biogenesis.