RESUMO
BACKGROUND: We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal. PURPOSE: The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability. METHODS: Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy. RESULTS: HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells. CONCLUSION: The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous micro-circulation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells.
Assuntos
Materiais Biocompatíveis/farmacologia , Fígado/metabolismo , Nanodiamantes/química , Animais , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imageamento Tridimensional , Cinética , Fígado/efeitos dos fármacos , Microscopia de Fluorescência , Tamanho da Partícula , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Endothelial lipase (EL), a member of the triglyceride lipase gene family, has been shown to be a key player in HDL metabolism. Northern blots revealed that EL was highly expressed in endothelium, thyroid, lung, placenta, liver, and testis. In liver and adrenal gland, EL protein was localized with vascular endothelial cells but not parenchymal cells. EL was shown to be upregulated in tissues such as atherosclerotic plaque where it was located in macrophages, endothelial cells, and medial smooth muscle cells. The purpose of this study was to investigate the cellular localization of EL in thyroid and other tissues where EL is known to be expressed. Besides its presence in vascular endothelial and smooth muscle cells, EL protein was detected in the epithelial cells that line the follicles within the thyroid gland. EL-specific immunostaining was also found near the cell surface as well as in the cytoplasm of adipocytes. Using immunoblots, EL expression was confirmed in cultured human omental and subcutaneous adipocytes. EL expression, however, was not found in preadipocytes. These findings suggest that EL plays a role in thyroid and adipocyte biology in addition to its well-known role in endothelial function and HDL metabolism.
Assuntos
Adipócitos/enzimologia , Células Epiteliais/enzimologia , Lipase/metabolismo , Glândula Tireoide/enzimologia , Animais , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Humanos , Imuno-Histoquímica , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Omento/citologia , Omento/enzimologia , Especificidade de Órgãos , Especificidade da Espécie , Gordura Subcutânea/citologia , Gordura Subcutânea/enzimologiaRESUMO
BACKGROUND: The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings. RESULTS: We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs. CONCLUSIONS: By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral , DNA/química , Herpesvirus Humano 4/fisiologia , Leucócitos Mononucleares/metabolismo , Linfócitos B/fisiologia , Doadores de Sangue , Linhagem Celular Transformada , Transformação Celular Viral/genética , DNA/metabolismo , Exoma/genética , Humanos , Análise de Sequência de DNARESUMO
Cannabinoids are known to be clinically beneficial for control of appetite disorders and nausea/vomiting, with emerging data that they can impact other GI disorders, such as inflammation. Post-inflammatory irritable bowel syndrome (PI-IBS) is a condition of perturbed intestinal function that occurs subsequent to earlier periods of intestinal inflammation. Cannabinoid 1 receptor (CB1R) and CB2R alterations in GI inflammation have been demonstrated in both animal models and clinically, but their continuing role in the post-inflammatory period has only been implicated to date. Therefore, to provide direct evidence for CBR involvement in altered GI functions in the absence of overt inflammation, we used a model of enhanced upper GI transit that persists for up to 4 weeks after a single insult by intracolonic 0.5% oil of mustard (OM) in mice. In mice administered OM, CB1R immunostaining in the myenteric plexus was reduced at day 7, when colonic inflammation is subsiding, and then increased at 28 days, compared to tissue from age-matched vehicle-treated mice. In the lamina propria CB2R immunostaining density was also increased at day 28. In mice tested 28 day after OM, either a CB1R-selective agonist, ACEA (1 and 3 mg/kg, s.c.) or a CB2R-selective agonist, JWH-133 (3 and 10 mg/kg, s.c.) reduced the enhanced small intestinal transit in a dose-related manner. Doses of ACEA and JWH-133 (1 mg/kg), alone or combined, reduced small intestinal transit of OM-treated mice to a greater extent than control mice. Thus, in this post-colonic inflammation model, both CBR subtypes are up-regulated and there is increased efficacy of both CB1R and CB2R agonists. We conclude that CBR remodeling occurs not only during GI inflammation but continues during the recovery phase. Thus, either CB1R- or CB2-selective agonists could be efficacious for modulating GI motility in individuals experiencing diarrhea-predominant PI-IBS.
RESUMO
Matriptase (membrane-type serine proteinase) was reported to play a role in nonmelanoma skin cancer progression. Moreover, it was shown to stimulate proteinase-activated receptor-2 (PAR(2)) in vitro. Hepatocyte growth factor activator inhibitor-1 (HAI-1), the matriptase inhibitor, is an important regulator of enzyme activity. Therefore, the aim of this study was to elucidate the putative role of matriptase, HAI-1, and PAR(2) in normal human skin, as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). In normal human epidermis, PAR(2) colocalized with matriptase and HAI-1. Immunoreactivity of all proteins was found to be diminished in BCCs. Likewise, PAR(2) immunoreactivity was significantly decreased, whereas matriptase immunoreactivity was enhanced with SCC progression. We could also show that matriptase was complexed to HAI-1 in normal human skin, whereas in SCCs, the enzyme was present in an unassociated form. Both a specific peptide agonist for PAR(2) and the proteinase domain of matriptase were able to induce intracellular calcium mobilization and inhibition of proliferation in cultured HaCaT keratinocytes. In conclusion, our results suggest that PAR(2) is a substrate for matriptase in human skin in vivo. Deregulation of these proteins delineates SCC progression.
Assuntos
Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/patologia , Divisão Celular/fisiologia , Linhagem Celular , Epiderme/metabolismo , Epiderme/patologia , Feminino , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.
Assuntos
Cistite/genética , Cistite/metabolismo , Regulação da Expressão Gênica , Receptor PAR-1/metabolismo , Animais , Antígenos/imunologia , Cálcio/metabolismo , Cromatina/metabolismo , Cistite/induzido quimicamente , Cistite/imunologia , Feminino , Expressão Gênica , Genoma , Imunoprecipitação , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A/metabolismo , Reação em Cadeia da Polimerase/métodos , Receptor PAR-1/deficiência , Frações Subcelulares/metabolismo , Substância P , Bexiga Urinária/metabolismoRESUMO
BACKGROUND: In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. RESULTS: Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. CONCLUSION: Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.
Assuntos
Cistite/metabolismo , Receptor PAR-1/metabolismo , Animais , Antígenos/imunologia , Linhagem Celular , Cistite/induzido quimicamente , Cistite/imunologia , Cistite/patologia , Edema/induzido quimicamente , Granulócitos/patologia , Humanos , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor PAR-1/agonistas , Receptor PAR-1/deficiência , Receptor PAR-2/efeitos dos fármacos , Receptor PAR-2/metabolismo , Receptores Ativados por Proteinase/metabolismo , Substância P , Bexiga Urinária/metabolismo , Urotélio/citologia , Urotélio/metabolismoRESUMO
Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.
Assuntos
DNA Recombinante , Células Epiteliais/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Epiteliais/citologia , Éxons/genética , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição GênicaRESUMO
Biomarkers in the clinical oncology field can have tremendous therapeutic impact especially if the marker is detected before clinical symptoms. This impact can be extended to the evaluation of clinical oncology treatments allowing evaluation of potential compounds to determine their efficacy in the disease treatment. The discovery of clinical biomarkers can consume time, resources and costs. Therefore, it is important that the most effective strategies are employed to discover these biomarkers. These strategies may include the integration of available genomic, proteomic and histopathological technologies, which could reduce the costs and aid in the validation of the biomarker. Certainly the type of biomarker needed to address a particularly defined problem will drive the type of technology. However, a single biomarker to diagnose a specific cancer can be as elusive as relying on a single technology. This review examines some of the technologies used to discover biomarkers and presents the use of combinatorial technical synergies to discover and validate potential clinical oncology biomarkers.
Assuntos
Biomarcadores Tumorais , Avaliação Pré-Clínica de Medicamentos/métodos , Genômica/métodos , Histocitoquímica/métodos , Humanos , Proteômica/métodosRESUMO
With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cgamma1 (PLCgamma1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCgamma as a downstream signaling partner, these methods may also benefit other drug discovery targets.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Indanos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Animais , Anticorpos Fosfo-Específicos/imunologia , Antineoplásicos/química , Neoplasias do Colo/enzimologia , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Indanos/química , Camundongos , Fosfolipase C gama , Fosforilação , Inibidores de Proteínas Quinases/química , Pirazóis/química , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.
Assuntos
Neoplasias/enzimologia , Diester Fosfórico Hidrolases/biossíntese , 3',5'-GMP Cíclico Fosfodiesterases , Células Endoteliais/enzimologia , Células Epiteliais/enzimologia , Humanos , Imuno-Histoquímica , Miócitos de Músculo Liso/enzimologia , Especificidade de ÓrgãosRESUMO
Activity-dependent neurotrophic factor (ADNF) is a novel, femtomolar-acting, glial-derived polypeptide (14 kDa) known to protect neurons from a variety of toxic insults. The active site for ADNF function is localized to a 9-amino-acid stretch (SALLRSIPA; ADNF-9). A few years later, a novel ADNF-9-like active peptide (NAPVSIPQ or NAP) was identified and shown to be expressed in the CNS and exhibit an activity profile similar to ADNF-9. Such studies suggest that ADNF-9 and NAP might function like other known neurotrophins and play a role in neural development and maintenance. The purpose of the present studies was to determine if ADNF-9 or NAP affects neurite outgrowth and synaptogenesis in rat hippocampal and cortical cultures. Using MAP2-FITC immunofluorescent labeling, we found that ADNF-9 and NAP promoted neurite outgrowth in a concentration-dependent manner, with maximal activity observed at femtomolar concentrations. Both peptides stimulated robust outgrowth in hippocampal cells (approximately 150% of control; p < 0.01) with a modest effect on cortical cells (approximately 20% of control; p < 0.05) similar to other known growth factors. However, the outgrowth-promoting effect was abolished in the absence of serum, suggesting that soluble factors might be necessary for the neurotrophic activity. Finally, we found that ADNF-9 and NAP increased synaptophysin expression in both rat hippocampal and cortical cultures. These results suggest that ADNF-9 and NAP might contribute to neuronal plasticity associated with development and repair after injury.
Assuntos
Proteínas do Tecido Nervoso/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/fisiologia , Oligopeptídeos/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Hipocampo/citologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Gravidez , Ratos , Sinaptofisina/metabolismoRESUMO
Oil of mustard (OM) is a potent neuronal activator that promotes allodynia and hyperalgesia within minutes of application. In this study, OM was used to induce an acute colitis. We also investigated whether intracolonic OM-induced inflammation alters gastrointestinal (GI) function over a longer time frame as a model of postinflammatory irritable bowel syndrome (PI-IBS). Mice given a single administration of 0.5% OM developed a severe colitis that peaked at day 3, was reduced at day 7, and was absent by day 14. At the peak response, there was body weight loss, colon shrinkage, thickening and weight increases, distension of the proximal colon, and diarrhea. Macroscopic inspection of the distal colon revealed a discontinuous pattern of inflammatory damage and occasional transmural ulceration. Histological examination showed loss of epithelium, an inflammatory infiltrate, destruction of mucosal architecture, edema, and loss of circular smooth muscle architecture. OM administration increased transit of a carmine dye bolus from 58% of the total length of the upper GI tract in untreated age-matched controls to as high as 74% when tested at day 28 post-OM. Mice in the latter group demonstrated a significantly more sensitive response to inhibition of upper GI transit by the mu-opioid receptor agonist loperamide compared with normal mice. OM induces a rapid, acute, and transient colitis and, in the longer term, functional changes in motility that are observed when there is no gross inflammation and thereby is a model of functional bowel disorders that mimic aspects of PI-IBS in humans.
Assuntos
Colite/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Síndrome do Intestino Irritável/fisiopatologia , Extratos Vegetais/efeitos adversos , Doença Aguda , Animais , Antidiarreicos/farmacologia , Colite/veterinária , Colo/imunologia , Colo/patologia , Diarreia/etiologia , Modelos Animais de Doenças , Inflamação , Intestino Grosso/fisiologia , Intestino Delgado/fisiologia , Síndrome do Intestino Irritável/veterinária , Loperamida/farmacologia , Masculino , Camundongos , Mostardeira , Extratos Vegetais/administração & dosagem , Óleos de Plantas , Úlcera/patologiaRESUMO
A spatial association between mast cells and nerves has been described in both the gastrointestinal and genitourinary tracts. However, the factors that influence the anatomic relationship between mast cells and nerves have not been completely defined. It has been suggested that the high-affinity receptor for substance P [neurokinin-1 (NK1)] might modulate this interaction. We therefore assessed mast cell-nerve relationships in tissues isolated from wild-type and NK1 receptor knockout (NK1-/-) mice. We now report that, in the complete absence of NK1 receptor expression, there is a significant increase in the number of mast cells without a change in the anatomic relationship between mast cell and nerves in stomach and bladder tissues at the light microscopic level. We next determined whether transplanted mast cells would maintain their spatial distribution, number, and contact with nerve elements. For this purpose, mast cell-deficient Kit(W)/Kit(W-v) mice were reconstituted with wild-type or NK1-/- bone marrow. No differences in mast cell-nerve contact were observed. These results suggest that NK1 receptor expression is important in the regulation of the number of mast cells but is not important in the interaction between mast cells and nerves. Furthermore, the interaction between mast cells and nerves is not mediated through NK1 receptor expression on the mast cell. Further studies are needed to determine the molecular pathway involved in mast cell migration and interaction with nerve elements, but the model of reconstitution of Kit(W)/Kit(W-v) mice with mast cells derived from different genetically engineered mice is a useful approach to further explore these mechanisms.
Assuntos
Mastócitos/fisiologia , Neurônios/fisiologia , Receptores da Neurocinina-1/metabolismo , Animais , Transplante de Medula Óssea , Contagem de Células , Feminino , Expressão Gênica , Camundongos , Camundongos Knockout , Estômago/anatomia & histologia , Estômago/inervação , Bexiga Urinária/anatomia & histologia , Bexiga Urinária/inervaçãoRESUMO
Deleted in malignant brain tumors 1 (DMBT1) is a candidate suppressor of malignancies of the brain, lung, gut, and breast. We have been studying gene expression in the uterus in the presence of estrogens and their antagonists. Here, we show that DMBT1 RNA levels are robustly increased by estrogen treatment in the uteri of ovariectomized monkeys and rats. In monkeys, the progestin antagonist mifepristone inhibits estrogen-dependent uterine proliferation. As determined by a microarray experiment and quantitative analysis of RNA levels, mifepristone inhibited estrogenic induction of DMBT1. DMBT1 was not expressed in intact monkeys that were treated with a gonadotropin agonist to suppress steroidogenesis. An in vitro transfection study with human DMBT1 promoter constructs showed that an Alu site approximately 3000 nucleotides upstream of the gene mediates estrogenic regulation. Surprisingly, the estrogen antagonists tamoxifen, raloxifene, and ICI 182,780 also induced gene expression via this Alu site. Rodents represent a more convenient model system for studying uterine biology than monkeys. In rats, uterine DMBT1 RNA levels were dramatically up-regulated by estrogen. Consistent with the transfection study, tamoxifen and raloxifene increased DMBT1 RNA levels in vivo, but ICI 182,780 inhibited an estrogen-induced increase. Immunohistochemical studies showed that DMBT1 is specifically induced in glandular and luminal epithelia of the rat endometrium. Our experiments establish that DMBT1 is an estrogen-responsive gene with a possible role in endometrial proliferation or differentiation, and they have implications for the putative tumor suppressive and mucosal protective functions of DMBT1 in the uterus.
Assuntos
Aglutininas/fisiologia , Endométrio/metabolismo , Epitélio/metabolismo , Estradiol/análogos & derivados , Estrogênios/metabolismo , Regulação da Expressão Gênica , Mucinas/fisiologia , Receptores de Superfície Celular/fisiologia , Elementos Alu , Animais , Northern Blotting , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Haplorrinos , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , Mucinas/biossíntese , Mucosa/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/química , RNA/metabolismo , Cloridrato de Raloxifeno/farmacologia , Ratos , Ratos Sprague-Dawley , Tamoxifeno/farmacologia , Transfecção , Proteínas Supressoras de Tumor , Regulação para Cima , Útero/metabolismoRESUMO
Inhibition of angiogenesis may have wide use in the treatment of cancer; however, this approach alone will not cause tumor regression but may only slow the growth of solid tumors. The clinical potential of antiangiogenic agents may be increased by combining them with conventional chemotherapeutics. 4-[4-(1-Amino-1-methylethyl)phenyl]-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile (JNJ-17029259) represents a novel structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2) and other tyrosine kinases involved in angiogenesis, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, VEGF-R1, and VEGF-R3, but have little activity on other kinase families. At nanomolar levels, JNJ-17029259 blocks VEGF-stimulated mitogen-activated protein kinase signaling, proliferation/migration, and VEGF-R2 phosphorylation in human endothelial cells; inhibits the formation of vascular sprouting in the rat aortic ring model of angiogenesis; and interferes with the development of new veins and arteries in the chorioallantoic membrane assay. At higher concentrations of 1 to 3 microM, this compound shows antiproliferative activity on cells that may contribute to its antitumor effects. JNJ-17029259 delays the growth of a wide range of human tumor xenografts in nude mice when administered orally as single-agent therapy. Histological examination revealed that the tumors have evidence of reduced vascularity after treatment. In addition, JNJ-17029259 enhances the effects of the conventional chemotherapeutic drugs doxorubicin and paclitaxel in xenograft models when administered orally in combination therapy. An orally available angiogenesis inhibitor that can be used in conjunction with standard chemotherapeutic agents to augment their activity may have therapeutic benefit in stopping the progression of cancer and preventing metastasis.
Assuntos
Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Nitrilas/uso terapêutico , Paclitaxel/uso terapêutico , Pirimidinas/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimioterapia Combinada , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Camundongos , Nitrilas/farmacologia , Pirimidinas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.
Assuntos
Cromossomos Humanos Par 16 , Macrófagos/enzimologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Colo/enzimologia , Primers do DNA , Humanos , Pulmão/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Baço/enzimologia , Células U937RESUMO
Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.
Assuntos
Aciltransferases/metabolismo , Fibroblastos/metabolismo , Neoplasias/metabolismo , Divisão Celular , Linhagem Celular Transformada , Humanos , Imuno-Histoquímica/métodos , Recém-Nascido , Subunidades Proteicas , Serina C-PalmitoiltransferaseRESUMO
Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists of at least two subunits, SPT1 and SPT2. In this study we analyzed the expression of SPT1 and SPT2 in normal human tissues. Strong SPT1 and SPT2 expression was observed in pyramidal neurons in the brain, in colon epithelium, and in mucosal macrophages. However, SPT2 expression was more prominent than SPT1 in the colon mucosal macrophages, the adrenomedullary chromaffin cells and endothelium, and in the uterine endothelium. SPT2 was localized in both nuclei and cytoplasm of the adrenomedullary chromaffin cells, whereas SPT1 was primarily cytoplasmic. These observations link enhanced SPT expression to proliferating cells, such as the lung, stomach, intestinal epithelium, and renal proximal tubular epithelium, and to potentially activated cells such as neurons, chromaffin cells, and mucosal macrophages. A baseline expression of SPT, established by this study, may serve as a measure for aberrant expression in various disease states.