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OBJECTIVE: To study the chemical constituents of Phyllanthus emblica. METHODS: The chemical constituents were isolated and purified by silica gel, polyamide and Sephadex LH-20 chromatography. Their structures were elucidated by physicochemical proper- ties and spectral analysis. RESULTS: 13 compounds were isolated and identified as Triacontanol (1), Triacontanoic acid (2), ß-Amyrin ke- tone (3), Betulonic acid (4), Daucosterol (5), Lupeol acetate (6), ß-Amyrin-3-palmitate (7), Gallic acid (8), Betulinic acid (9), Ursolic acid (10), Oleanolic acid (11), Quercetin (12) and Rutin (13). CONCLUSION: Compounds 1,2,4,6,7,9,10 and 11 are obtained from Phyllanthus emblica for the first time.
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Phyllanthus emblica/química , Compostos Fitoquímicos/química , Plantas Medicinais/química , Ácido Gálico , Ácido Oleanólico/análogos & derivados , Triterpenos Pentacíclicos , Compostos Fitoquímicos/isolamento & purificação , Quercetina , Rutina , Triterpenos , Ácido Betulínico , Ácido UrsólicoRESUMO
Although studies have revealed the significant impact of dietary fiber on growth performance and nutrient digestibility, the specific characteristics of the intestinal microbiota and gene regulation in pigs capable of digesting high-fiber diets remained unclear. To investigate the traits associated with roughage tolerance in the Chinese indigenous pig breed, we conducted comparative analysis of growth performance, apparent fiber digestibility, intestinal microbiota, SCFA concentrations and intestinal transcriptome in Tunchang pigs, feeding them diets with different wheat bran levels. The results indicated that the growth performance of Tunchang pigs was not significantly impacted, and the apparent total tract digestibility of crude fiber was significantly improved with increasing dietary fiber content. High-fiber diets altered the diversity of intestinal microbiota, and increased the relative abundance of Prevotella, CF231, as well as the concentrations of isobutyrate, valerate and isovalerate. The LDA analysis identified potential microbial biomarkers that could be associated with roughage tolerance, such as Prevotella stercorea, and Eubacterium biforme. In addition, appropriate high-fiber diets containing 4.34% crude fiber upregulated the mRNA expressions of PYY, AQP8, and SLC5A8, while downregulating the mRNA expressions of CKM and CNN1.This indicated that appropriate high-fiber diets may inhibit intestine motility and increase the absorption of water and SCFAs.
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Introduction: Hybridization has been widely used among Chinese wild boars to improve their growth performance and maintain meat quality. Most studies have focused on the genetic basis for such variation. However, the differences in the gut environment between hybrid and purebred boars, which can have significant impacts on their health and productivity, have been poorly understood. Methods: In the current study, metagenomics was used to detect the gut microbial diversity and composition in hybrid Batun (BT, Berkshire × Tunchang) pigs and purebred Tunchang (TC) pigs. Additionally, untargeted metabolomic analysis was used to detect differences in gut metabolic pathways. Furthermore, multiple molecular experiments were conducted to demonstrate differences in intestinal functions. Results: As a result of hybridization in TC pigs, a microbial change was observed, especially in Prevotella and Lactobacillus. Significant differences were found in gut metabolites, including fatty acyls, steroids, and steroid derivatives. Furthermore, the function of the intestinal barrier was decreased by hybridization, while the function of nutrient metabolism was increased. Discussion: Evidences were shown that hybridization changed the gut microbiome, gut metabolome, and intestinal functions of TC pigs. These findings supported our hypothesis that hybridization altered the gut microbial composition, thereby modifying the intestinal functions, even the host phenotypes. Overall, our study highlights the importance of considering the gut microbiome as a key factor in the evaluation of animal health and productivity, particularly in the context of genetic selection and breeding programs.
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OBJECTIVE: To investigate connective tissue growth factor (CTGF) expression before and after pulsed dye laser (PDL, 595 nm) treatment, and to better understand the mechanism of PDL treatment of keloids. METHOD: Twenty-six patients with keloids were recruited for this study. For each patient, two keloids of similar anatomic location, duration, texture, and appearance were chosen for study; one of these keloids was treated and the other served as a control. Three sessions of PDL treatment, with pulse duration of 1.5 milliseconds, spot size 7 mm, DCD duration 20 milliseconds/delay 10 milliseconds and fluence of 10 J/cm(2), were performed on the keloids at 3- to 4-week intervals. Punch biopsies were performed both on the treated and untreated keloids prior to the first treatment and after the final treatment. The specimens underwent realtime polymerase chain reaction (PCR) and immunohistochemistry (IHC) to investigate the CTGF mRNA and protein expression after PDL treatment. RESULTS: According to realtime PCR, the CTGF mRNA was significantly down-regulated after PDL treatment in 80.77% of patients as compared to the control group. IHC investigation showed that after treatment the CTGF positive cells also significantly decreased in number as compared to the control group in 80.77% of patients. Using the Vancouver scar scale (VSS), there was an average decrease of 20.85 ± 12.33% after PDL treatment. CONCLUSIONS: Pulsed dye laser treatment of keloids significantly down-regulates the expression of CTGF in most cases. This may partially explain the mechanism of action of PDL treatment of keloids.
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Fator de Crescimento do Tecido Conjuntivo/metabolismo , Queloide/cirurgia , Lasers de Corante/uso terapêutico , Adulto , Biomarcadores/metabolismo , Regulação para Baixo , Feminino , Humanos , Imuno-Histoquímica , Queloide/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Resultado do TratamentoRESUMO
PURPOSE: How herpes simplex virus (HSV) is transported from the infected neuron cell body to the axon terminal is poorly understood. Several viral proteins are candidates for regulating the process, but the evidence is controversial. We compared the results of Us9 deletions in two HSV strains (F and NS) using a novel quantitative assay to test the hypothesis that the viral protein Us9 regulates the delivery of viral DNA to the distal axon of retinal ganglion cells in vivo. We also deleted a nine-amino acid motif in the Us9 protein of F strain (Us9-30) to define the role of this domain in DNA delivery. METHODS: The vitreous chambers of murine eyes were infected with equivalent amounts of F or NS strains of HSV. At 3, 4, or 5 days post infection (dpi), both optic tracts (OT) were dissected and viral genome was quantified by qPCR. RESULTS: At 3 dpi, the F strain Us9- and Us9-30 mutants delivered less than 10% and 1%, respectively, of the viral DNA delivered after infection with the Us9R (control) strain. By 4 and 5 dpi, delivery of viral DNA had only partially recovered. Deletion of Us9 in NS-infected mice has a less obvious effect on delivery of new viral DNA to the distal OT. By 3 dpi the NS Us9-strain delivered 22% of the DNA that was delivered by the NS wt, and by 4 and 5 dpi the amount of Us9-viral DNA was 96% and 81%, respectively. CONCLUSIONS: A highly conserved acidic cluster within the Us9 protein plays a critical role for genome transport to the distal axon. The transport is less dependent on Us9 expression in the NS than in the F strain virus. This assay can be used to compare transport efficiency in other neurotropic viral strains.
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Axônios/virologia , DNA Viral/genética , Regulação Viral da Expressão Gênica , Células Ganglionares da Retina/virologia , Simplexvirus/genética , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Animais , Axônios/metabolismo , Axônios/patologia , Linhagem Celular , Modelos Animais de Doenças , Infecções Oculares Virais/genética , Infecções Oculares Virais/patologia , Infecções Oculares Virais/virologia , Genoma Viral , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Proteínas do Envelope Viral/biossíntese , Proteínas Virais/metabolismoRESUMO
UNLABELLED: The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers. SIGNIFICANCE: This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.
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Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Transativadores/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3 , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleosídeos/farmacologia , Análise de Sobrevida , Transativadores/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes. RESULTS: In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor. CONCLUSION: These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.
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Carcinoma/genética , Ordem dos Genes , Genes Neoplásicos , Genoma Humano , Linhagem Celular Tumoral , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Quebras de DNA , Biblioteca Gênica , Humanos , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Análise de Sequência de DNA , Transcrição GênicaRESUMO
The transcription factor ZNF217 is often amplified in ovarian cancer, but its role in neoplastic progression is unknown. We introduced ZNF217-HA by adenoviral and retroviral infection into normal human ovarian surface epithelial cells (OSE), i.e., the source of ovarian cancer, and into SV40 Tag/tag expressing, p53/pRB-deficient OSE with extended but finite life spans (IOSE). In OSE, ZNF217-HA reduced cell-substratum adhesion and accelerated loss of senescent cells, but caused no obvious proneoplastic changes. In contrast, ZNF217-HA transduction into IOSE yielded two permanent lines, I-80RZ and I-144RZ, which exhibited telomerase activity, stable telomere lengths, anchorage independence and reduced serum dependence, but were not tumorigenic in SCID mice. This immortalization required short-term EGF treatment near the time of crisis. The permanent lines were EGF-independent, but ZNF217-dependent since siRNA to ZNF217 inhibited anchorage independence and arrested growth. Array CGH revealed genomic changes resembling those of ovarian carcinomas, such as amplicons at 3q and 20q, and deletions at 4q and 18, associated with underexpressed annexin A10, N-cadherin, desmocollin 3 and PAI-2, which have been reported as tumor suppressors. The lines overexpressed EEF1A2, SMARA3 and STAT1 and underexpressed other oncogenes, tumor suppressors and extracellular matrix/adhesion genes. The results implicate ZNF217 as an ovarian oncogene, which is detrimental to senescing normal OSE cells but contributes to neoplastic progression in OSE with inactivated p53/RB. The resemblance of the genomic changes in the ZNF217-overexpressing lines to ovarian carcinomas provides a unique model to investigate interrelationships between these changes and ovarian neoplastic phenotypes.
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Neoplasias Ovarianas/etiologia , Transativadores/fisiologia , Adenoviridae/genética , Células Cultivadas , Progressão da Doença , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Hidrocortisona/farmacologia , Hibridização de Ácido Nucleico , Neoplasias Ovarianas/patologia , Fenótipo , Transativadores/genéticaRESUMO
Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.
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Fosfatidilinositol 3-Quinases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Dimerização , Camundongos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and co-amplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes anti-apoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project.
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Neoplasias da Mama/genética , Análise de Sequência de DNA/métodos , Transcrição Gênica , Linhagem Celular Tumoral , Cromossomos Artificiais Bacterianos/metabolismo , Cromossomos Humanos , Feminino , Perfilação da Expressão Gênica/métodos , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
Chromosome 20q13.2 is amplified in 20-30% of early-stage breast tumors and is associated with poor prognosis. Detailed mapping of the amplified region using molecular cytogenetics, positional cloning and genomic sequencing culminated in a detailed molecular description of the candidate oncogene ZNF217. ZNF217 proteins resemble Kruppel-like transcription factors, localize predominately to the nucleus and associate with proteins involved in transcriptional repression. The findings that ZNF217 can immortalize human mammary epithelial cells and that its amplification is associated with poor prognosis suggest that it may play roles in both early- and late-stage breast cancer. We present evidence that ZNF217 can attenuate apoptotic signals resulting from telomere dysfunction as well as from doxorubicin-induced DNA damage and that silencing ZNF217 with siRNA restores sensitivity to doxorubicin. Moreover, elevated ZNF217 leads to increased phosphorylation of Akt, whereas inhibition of the phosphatidylinositol 3 kinase pathway and Akt phosphorylation decreases ZNF217 protein levels and increases sensitivity to doxorubicin. These results suggest that ZNF217 may promote neoplastic transformation by increasing cell survival during telomeric crisis and may promote later stages of malignancy by increasing cell survival during chemotherapy.
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Apoptose/genética , Neoplasias da Mama/genética , Cromossomos Humanos Par 20/genética , Proteínas de Neoplasias/metabolismo , Telômero/fisiologia , Transativadores/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Primers do DNA , Doxorrubicina , Tratamento Farmacológico , Inativação Gênica , Humanos , Immunoblotting , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Telômero/genética , Transativadores/genéticaRESUMO
In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via gamma-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.
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Ácido 1-Carboxiglutâmico/química , Membrana Celular/metabolismo , Vitamina K/química , Sequência de Aminoácidos , Animais , Arginina/química , Sítios de Ligação , Coagulação Sanguínea , Cálcio/metabolismo , Bovinos , Cristalografia por Raios X , Íons , Lisofosfolipídeos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Protrombina/químicaRESUMO
Genome rearrangements are important in evolution, cancer, and other diseases. Precise mapping of the rearrangements is essential for identification of the involved genes, and many techniques have been developed for this purpose. We show here that end-sequence profiling (ESP) is particularly well suited to this purpose. ESP is accomplished by constructing a bacterial artificial chromosome (BAC) library from a test genome, measuring BAC end sequences, and mapping end-sequence pairs onto the normal genome sequence. Plots of BAC end-sequences density identify copy number abnormalities at high resolution. BACs spanning structural aberrations have end pairs that map abnormally far apart on the normal genome sequence. These pairs can then be sequenced to determine the involved genes and breakpoint sequences. ESP analysis of the breast cancer cell line MCF-7 demonstrated its utility for analysis of complex genomes. End sequencing of approximately 8,000 clones (0.37-fold haploid genome clonal coverage) produced a comprehensive genome copy number map of the MCF-7 genome at better than 300-kb resolution and identified 381 genome breakpoints, a subset of which was verified by fluorescence in situ hybridization mapping and sequencing.