RESUMO
PURPOSE: Undesired drug sorption on laboratory material surfaces reduces the performance of analytical methods and results in the generation of unreliable data. Hence, we characterized the sorption of drugs and evaluated the sorption extent using a linear free energy relationship (LFER) model with Abraham solvation parameters of drugs. Furthermore, to prevent sorption, the effects of additives, such as organic solvents and salts, were evaluated. METHODS: The sorption of fifteen model drugs (concentration: 2 µM), with various physicochemical properties, on materials in 0.2% dimethyl sulfoxide aqueous solutions was evaluated. Drug sorption extent on the materials was determined using high-performance liquid chromatography. The obtained results were analyzed using an LFER model with Abraham solvation parameters of the drugs. The effect of additives on the sorption of itraconazole, one of the most hydrophobic drugs among those tested in this study, was investigated. RESULTS: Sorption was dependent on the physicochemical properties of drugs, rather than the type of materials used, and additives altered the rate of drug sorption. Equations were developed to evaluate the sorption extent (nmol) of drugs to glass and polypropylene using the Abraham solvation parameters of the drugs. CONCLUSIONS: LFER modeling with Abraham solvation parameters of drugs enabled us to evaluate drug sorption on materials. All the additives altered the rate of drug sorption, and some organic solvents effectively prevented sorption. The developed LFER model would be useful for assessment of the sorption properties of compounds in in vitro evaluations in drug discovery research and various other biochemical fields.
Assuntos
Modelos Químicos , Compostos Orgânicos/química , Preparações Farmacêuticas/química , Adsorção , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Descoberta de Drogas , Solventes , TermodinâmicaRESUMO
Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.
Assuntos
Ameloblastos/citologia , Linhagem da Célula , Separação Celular/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Porco Miniatura/embriologia , Germe de Dente/citologia , Ameloblastos/ultraestrutura , Animais , Células Cultivadas , Esmalte Dentário/citologia , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Glicogênio/metabolismo , Fenótipo , Vesículas Secretórias/metabolismo , SuínosRESUMO
A new cell line designated Nur-1 has been established from human endometrioid adenocarcinoma, Grade 1, pT1a, PN1 (3/24), Stage IIIc (International Federation of Gynecology and Obstetrics/Union for International Cancer Control (FIGO/UICC TNM Classification of Malignant Tumours, 7th ed.). Cytological findings of Nur-1 cells reveal anaplastic and pleomorphic features such as anisonucleosis, nucleolar pleomorphism, and piling-up tendency in cellular arrangement. Distribution of the chromosome number is found at the hyperploid range, and the apparent marker chromosome has not been identified. The original tumor and graft of the Nur-1 cell line had a large amount of estrogen receptors and progesterone receptors, as revealed by immunohistochemistry. The cytokeratin pattern of the tumor was positive for cytokeratin-7 and negative for cytokeratin-20. However, a few cells were positive for cytokeratin-20 in the original tumor. Nur-1 cells express mRNA of estrogen receptors and progesterone receptors, cytokeratin-7, and cytokeratin-20 at 105 passages. These findings are consistent with the cytokeratin pattern of endometrial glandular cells. The cells make contact with each other via interdigitation and desmosomes. They possess bundles of microtubules and tonofilaments and many free ribosomes. Some cells have various sizes of phagosomes. The Nur-1 cell line exceeded 102 passages in 5 years, and multiplication of the cells is stable. The modal number of the Nur-1 cell line is 91-92 (56 %). The Nur-1 cells develop well-differentiated adenocarcinoma in tumors sustained in nude mice that resemble the original tumors.
Assuntos
Carcinoma Endometrioide , Neoplasias Uterinas , Animais , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromossomos Humanos/genética , Desmossomos , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Queratina-7/metabolismo , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fagossomos , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.
Assuntos
Células-Tronco Adultas/transplante , Polpa Dentária/citologia , Hepatócitos/citologia , Hepatócitos/transplante , Cirrose Hepática/terapia , Doença Aguda , Células-Tronco Adultas/citologia , Animais , Técnicas de Cultura Celular por Lotes/métodos , Diferenciação Celular , Células Cultivadas , Humanos , Cirrose Hepática/patologia , Masculino , Ratos Nus , Engenharia Tecidual/métodos , Esfoliação de Dente , Resultado do TratamentoRESUMO
The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.
Assuntos
Diferenciação Celular/genética , Células Epiteliais/citologia , Células Musculares/citologia , Ligamento Periodontal/citologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Células Musculares/metabolismo , Ligamento Periodontal/metabolismo , Periodonto/citologia , Ratos , Regeneração , Células-Tronco/citologia , Dente/citologia , CicatrizaçãoRESUMO
Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Rabdomiossarcoma/patologia , Neoplasias da Língua/patologia , Actinas/análise , Adolescente , Animais , Linhagem Celular Tumoral , Desmina/análise , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia Eletrônica , Mitose , Miogenina/análise , Miosinas/análise , Rabdomiossarcoma/genética , Neoplasias da Língua/genética , Translocação Genética , Transplante Heterólogo , Troponina T/análiseRESUMO
Abstract Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.
Assuntos
Melanoma/patologia , Neoplasias Bucais/patologia , Idoso , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Bochecha , Meios de Cultura , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Melaninas/biossíntese , Melanoma/genética , Melanoma/metabolismo , Melanoma/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Mucosa Bucal , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/ultraestrutura , Transplante de NeoplasiasRESUMO
According to recent genome-wide association studies, a number of single nucleotide polymorphisms is reported to be associated with diseases or several clinical markers. Among them, adiponectin (ADIPOQ) and perilipin (PLIN) polymorphisms are major factors of obesity. However, the association between lifestyle factor, these polymorphisms and obesity-related clinical markers in Japanese is not well researched. Therefore, the aim of present study is to investigate the association between lifestyle factor, polymorphisms of lipid metabolic genes, and clinical markers in 148 middle-aged Japanese males. The study revealed that ADIPOQ 45 T>G and ADIPOQ 276 G>T genotypes were significantly associated with triglyceride, total cholesterol, hemoglobin A1c (HbA1c) in blood and body mass index (BMI). PLIN4 11482 G>A and hormone sensitive lipase (LIPE)-60 C>G genotypes were respectively associated with BMI and serum triglyceride. Not only genetic factors but also lifestyle factors influence several clinical markers. The BMI of subjects who like sweets and have the GG allele in ADIPOQ 276 G>T was higher than that of subjects who don't like sweets. The habit of eating fruits and fish affected low-density lipoprotein-cholesterol of the GT allele and HbA1c of the TT allele in ADIPOQ 276 G>T. Those findings indicate improvement and conservation of lifestyle depending on genetic predisposition in ADIPOQ, PLIN and LIPE should be encouraged.
Assuntos
Adiponectina/genética , Estilo de Vida , Obesidade/sangue , Obesidade/genética , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único/genética , Esterol Esterase/genética , Adiponectina/sangue , Adulto , Biomarcadores/sangue , Índice de Massa Corporal , Proteínas de Transporte , Colesterol/sangue , Colesterol/genética , Comportamento Alimentar , Hemoglobinas Glicadas/genética , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Perilipina-1 , Fosfoproteínas/sangue , Fatores de Risco , Esterol Esterase/sangue , Triglicerídeos/sangue , Triglicerídeos/genéticaRESUMO
A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 degrees C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.
Assuntos
Antineoplásicos/farmacologia , Carcinossarcoma/patologia , Neoplasias Ovarianas/patologia , Animais , Antígenos de Neoplasias , Carcinossarcoma/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromossomos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Ovarianas/genéticaRESUMO
Chlamydomonas acidophila KT-1 and Chlamydomonas acidophila DVB238 exhibit a strong heavy metal tolerance, but C. acidophila DVB238 can accumulate a much higher amount of Cadmium (Cd) than C. acidophila KT-1. Phytochelatins (PCs) are known to play an important role in the detoxification of several toxic heavy metals, but the relationship between PCs and Cd accumulation is not clear. PC metabolism and Cd accumulation were investigated by using three Chlamydomonas strains including Chlamydomonas reinhardtii C-9 as a standard alga. The results showed that the PC content did not correlate closely with the level of Cd accumulation, maintenance of a high GSH level seeming to be more important for Cd accumulation. The ultrastructure of C. acidophila KT-1 was extremely disrupted by a great increase in starch granules, which resulted in a moribund state, but hyper-accumulator C. acidophila DVB238 did not exhibit an increase in starch granules in its cells, in spite of Cd accumulation in its chloroplasts, cytosol and vacuoles. These results indicated that C. acidophila DVB238 probably has a developed detoxification system preventing such as destruction of the cells due to Cd toxicity.
Assuntos
Cádmio/metabolismo , Chlamydomonas/metabolismo , Glutationa/metabolismo , Aminoaciltransferases/metabolismo , Animais , Cádmio/farmacologia , Chlamydomonas/efeitos dos fármacos , Chlamydomonas/ultraestrutura , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Glutamato-Cisteína Ligase/metabolismo , Inativação Metabólica , FitoquelatinasRESUMO
Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo (31)P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.