RESUMO
An equid herpesvirus 5 (EHV-5) infection was detected in lesioned skin from a nine-year-old Holsteiner stallion in the south of Germany. Macroscopically, the animal displayed a non-pruritic, multifocal, pustular dermatitis around both eyes, nostrils and the muzzle, which had been ongoing for one year. Histopathologically, skin lesions were characterized by orthokeratotic to parakeratotic hyperkeratosis, pustular dermatitis, epidermal hyperplasia, apoptotic keratinocytes, a lympho-plasmahistiocytic interface dermatitis with hydropic degeneration of keratinocytes, and perivascular to diffuse, lympho-histiocytic infiltrations. The stratum granulosum and the upper part of the stratum spinosum contained multiple amphophilic, intranuclear inclusion bodies. By in situ hybridization and immunohistochemistry herpesvirus DNA and protein, respectively, were detected within keratinocytes containing inclusion bodies. Sequencing of the PCR-product revealed the presence of EHV-5 DNA. This is the first description of a dermatitis associated with EHV-5 in a horse, indicating that EHV-5 should be considered as an etiology of lymphohistiocytic interface dermatitis with intranuclear inclusion bodies in horses and is similar to herpes-associated erythema multiforme in humans.
Assuntos
Dermatite/veterinária , Gammaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/veterinária , Doenças dos Cavalos/virologia , Animais , Dermatite/virologia , Diagnóstico Diferencial , Eritema Multiforme/patologia , Eritema Multiforme/virologia , Gammaherpesvirinae/genética , Alemanha , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Doenças dos Cavalos/patologia , Cavalos , Hibridização In Situ , Queratinócitos/virologia , Reação em Cadeia da Polimerase/veterináriaRESUMO
The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC(50) values of 1 and 0.2 microM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED(50) values of 1 and 0.1 microM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC(1) receptor agonists which inhibit MCF-7 cellular viability.
Assuntos
Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Elipticinas/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Sequência de Aminoácidos , Neoplasias da Mama/genética , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Receptores Tipo I de Polipeptídeo Intestinal VasoativoRESUMO
OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.
Assuntos
Antígenos Virais/análise , Doenças do Gato/virologia , DNA Viral/análise , Fibrossarcoma/veterinária , Papillomaviridae/isolamento & purificação , Vacinas contra Papillomavirus , Vacinação/veterinária , Vacinas Virais/efeitos adversos , Animais , Doenças do Gato/patologia , Gatos , DNA Viral/química , DNA Viral/isolamento & purificação , Fibrossarcoma/patologia , Fibrossarcoma/virologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Papillomaviridae/genética , Papillomaviridae/imunologia , Reação em Cadeia da Polimerase/veterinária , Análise de Sequência de DNA , Vacinação/efeitos adversosRESUMO
In principle, cell surface receptors that are overexpressed in tumor tissue could serve as targets for anticancer drugs attached to receptor ligands. The purpose of this paper is to identify the necessary elements for a successful receptor-targeted drug. We used the gastrin/cholecystokinin type B receptor as a model delivery system, and we report on the synthesis, trafficking, and in vitro and in vivo evaluation of heptagastrin, the C-terminal heptapeptide of gastrin, linked via an appropriate linker to a potently cytotoxic ellipticine derivative, 1-[3-[N-(3-aminopropyl)-N-methylamino]propyl]amino-9-methoxy-5, 11-dimethyl-6H-pyrido[4,3-b]carbazole. These data, and previous work from our laboratory, show that the drug-complexed ligand is sorted to lysosomes whereas the receptor is recycled to the plasma membrane. The lysosomal processing of the ligand/drug construct depends on the linker between the ligand sequence and the cytotoxic moiety. We show that heptagastrin linked to ellipticine via a succinoyl-substituted pentapeptide, AlaLeuAlaLeuAla, is at least 10(3) more toxic to cholecystokinin type B receptor-positive NIH/3T3 cells than to isogenic NIH/3T3 cells lacking the receptor. The conjugated drug eradicated all receptor-positive tumor cells in vivo without producing any general toxicity. The data indicate that the density of the cell surface receptor, the properties of the cytotoxic moiety, and the correct processing of the drug-conjugated ligand in lysosomes are crucial to the effectiveness of a receptor-targeted drug.
Assuntos
Antineoplásicos/farmacologia , Receptores da Colecistocinina/efeitos dos fármacos , Células 3T3 , Sequência de Aminoácidos , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Cálcio/metabolismo , Membrana Celular/metabolismo , Elipticinas/síntese química , Elipticinas/química , Elipticinas/farmacologia , Gastrinas/síntese química , Gastrinas/química , Gastrinas/farmacologia , Humanos , Cinética , Ligantes , Lisossomos/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Receptor de Colecistocinina B , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , TransfecçãoAssuntos
Ácido Aspártico Endopeptidases/metabolismo , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Catepsina D/metabolismo , Colecistocinina/metabolismo , Gastrinas/metabolismo , Hemoglobinas/metabolismo , Humanos , Dados de Sequência Molecular , Pepsina A/metabolismo , SuínosRESUMO
We have previously demonstrated that RGD peptides prevent tubular obstruction in ischemic acute renal failure (ARF) suggested that exposed unoccupied integrin receptors represent the target for such therapy. The present study investigated the topography of RGD binding sites and integrin receptors in ischemic rat kidneys. Two RGD peptides were synthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD peptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjected to 45 minutes of renal artery occlusion kidneys were harvested at different times post-ischemia, and stained with RGD peptides and a panel of antibodies to integrins. In control, Bt-RGD staining was undetectable in alkaline phosphatase histochemistry, whereas immunofluorescence detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP staining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed binding to the basolateral and apical aspects of proximal tubules, peritubular capillaries, and desquamated cells within tubular lumen. The most conspicuous staining of ischemic kidneys was obtained with antibodies to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and the alpha V (glomeruli, tubular epithelia, intima of blood vessels stained faintly, while the obstructing cellular conglomerates showed intense staining) subunits. Double staining with Bt-RGD and antibodies against the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro attachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding sites and beta 1 integrin subunits along the apical aspect of tubular epithelia and on the surface of desquamated cells is in concert with the hypothesis on the pathogenetic role of RGD-recognizing integrins in tubular obstruction. The expression of RGD binding sites along the intimal surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.
Assuntos
Isquemia/metabolismo , Rim/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Imuno-Histoquímica , Integrinas/metabolismo , Rim/irrigação sanguínea , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Microscopia de Fluorescência , Peptídeos/síntese química , Peptídeos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
A chimeric protein consisting of the cholecystokinin receptor type A (CCKAR) and the green fluorescent protein (GFP) was used for studying receptor localization, internalization, and recycling in live cells in real time in four different cell lines. Fusion of the C terminus of the CCKAR to the N terminus of the GFP did not alter receptor ligand binding affinity, signal transduction, or the pattern of receptor surface expression and receptor-mediated cholecystokinin (CCK) internalization. The use of a new GFP mutant with increased fluorescence allowed the continuous observation of CCKAR-GFP in stably expressing cell lines. Newly obtained biologically active fluorescent derivatives of CCK were used for simultaneous observation of receptor and ligand trafficking in CHO, NIH/3T3, and HeLa cells stably expressing the fluorescent CCKAR and in transiently transfected COS-1 cells. Receptor internalization was predominantly ligand dependent in HeLa, COS-1, and CHO cells, but was mostly constitutive in NIH/3T3 cells, suggesting the existence of cell-specific regulation of receptor internalization. The CCKAR antagonists, L-364,718 and CCK 27-32 amide potently inhibited spontaneous internalization of the receptor. The average sorting time of CCK and the receptor in the endosomes was about 25 min. The receptor recycled back to the cell membrane with an average time of 60 min. While the ligands sorted to lysosomes, no receptor molecules could be detected there, and no receptor degradation was observed during recycling. These results demonstrate the usefulness of GFP tagging for real time imaging of G protein-coupled receptor trafficking in living cells and suggest that this technique may be successfully applied to the study of the regulation and trafficking mechanisms of other receptors.
Assuntos
Endocitose , Fosfatos de Inositol/metabolismo , Receptores da Colecistocinina/fisiologia , Sincalida/farmacologia , Células 3T3 , Animais , Células CHO , Células COS , Cricetinae , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/biossíntese , Camundongos , Ratos , Receptor de Colecistocinina A , Receptores da Colecistocinina/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Sincalida/metabolismo , TransfecçãoRESUMO
Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4-7 min) and internalization of fluorescent ligand upon binding at room temperature and 37 degrees C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37 degrees C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.
Assuntos
Carcinoma de Células Acinares/metabolismo , Carcinoma/metabolismo , Endocitose , Gastrinas/metabolismo , Neoplasias Gastrointestinais/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores da Colecistocinina/metabolismo , Células 3T3 , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Cicloeximida/farmacologia , Endocitose/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Camundongos , Microscopia Confocal , Microscopia Imunoeletrônica , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , Transfecção , Transferrina/metabolismo , Células Tumorais CultivadasRESUMO
The reactions of calf thymus DNA with ten 1-(2-chloroethyl)-3-alkyl-3-acyltriazenes of varying acyl side chain structure were studied alone, or in the presence of porcine liver esterase in pH 7.0 phosphate buffer. In several of the key triazenes, the acyl substituent contained a free carboxylic acid group. With esterase present in the reaction mixture, the resultant levels of DNA alkylation could be correlated with the kinetic rates of decomposition of the triazenes. Under these conditions, the predominant pathway of decomposition involved deacylation of the parent triazene and eventual production of an alkanediazonium ion. This intermediate subsequently alkylated DNA--guanine to give 7-alkylguanine as the principal reaction product. In the absence of esterase, the order of DNA alkylation for all of the acyltriazenes did not correlate with their respective rates of decomposition, leading to the conclusion that the triazenes did not decompose by the expected mode of uncatalyzed N(2)-N(3) heterolyic cleavage. The major DNA alkylation product from the N(3)-methyltriazenes was 7-methylguanine, instead of the expected 7-(chloroethyl)- and 7-(hydroxyethyl)guanine products, which suggested that the acyl group was being hydrolyzed. However, acyltriazenes with an N(3)-benzyl group rather than a methyl in this position produced very little 7-benzylguanine product, contrary to prediction. An alternative mechanism involving internally assisted hydrolysis of the side chain ester is proposed to explain these results. NMR product analysis and computational studies were carried out to lend support to the postulated mechanism.
Assuntos
Alquilantes/farmacologia , DNA/metabolismo , Triazenos/farmacologia , Alquilantes/química , Alquilação , Animais , Bovinos , DNA/efeitos dos fármacos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Químicos , Timo , Triazenos/química , Triazenos/metabolismoRESUMO
The gastrin receptor is expressed in various human cancers, such as the adenocarcinoma of the colon. The peptide hormone gastrin and the C-terminal peptides derived from it act as growth factors for these cancers. The hypothesis for the present work was to use the gastrin receptor as a target for appropriately constructed cytotoxic agents. We developed methods to link tetragastrin and pentagastrin by their N-termini to cytotoxic 1-(2-chloroethyl)-3-benzyl-3-succinoyltriazene. These compounds, CBS-4 and CBS-5, respectively, whose complete structures were determined by multinuclear NMR and mass spectrometry, competed effectively with gastrin in an assay using either guinea pig stomach fundus or the rat acinar tumor cell line AR42J as the source of the receptor. CBS-5 was cytotoxic to AR42J cells but was not toxic to A549 human lung cancer cells, which do not express the receptor.