RESUMO
Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is greatly promoted by valproic acid (VPA) with cAMP elevating agents thorough NO signaling pathways, but its mechanism is not fully understood. In the present study, we investigate the involvement of protein S-nitrosylation in the VPA-promoted neuronal differentiation of ASCs. The whole amount of S-nitrosylated protein was increased by the treatment with VPA alone for three days in ASCs. An inhibitor of thioredoxin reductase (TrxR), auranofin, further increased the amount of S-nitrosylated protein and enhances the VPA-promoted neuronal differentiation in ASCs. On the contrary, another inhibitor of TrxR, dinitrochlorobenzene, inhibited the VPA-promoted neuronal differentiation in ASCs even with cAMP elevating agents, which was accompanied by unexpectedly decreased S-nitrosylated protein. It was considered from these results that increased protein S-nitrosylation is involved in VPA-promoted neuronal differentiation of ASCs. By the proteomic analysis of S-nitrosylated protein in VPA-treated ASCs, no identified proteins could be specifically related to VPA-promoted neuronal differentiation. The identified proteins, however, included those involved in the metabolism of substances regulating neuronal differentiation, such as aspartate and glutamate.
Assuntos
Neurônios , Ácido Valproico , Ácido Valproico/farmacologia , Neurônios/metabolismo , Proteômica , Células-Tronco/metabolismo , Tecido AdiposoRESUMO
Expression of desmin, an intermediate filament, in the myometrium and cervix were investigated in peripartum rats (full term day 22 of pregnancy (DP22)). Des mRNA was expressed in lesser amounts in the cervix at peripartum (DP17 and 21, and day of birth 1 (DB1)), compared to those in the cervixes of ovariectomized rats. Immunohistochemical analysis revealed that desmin protein was diffusely present in the myometrium, and locally in the epithelium of the cervix. Western blot analysis showed that desmin protein levels in the myometrium increased 4- to 6-fold at DP17, 21 and DB1, and decreased rapidly at DB2 to the basal level observed in ovariectomized or non-pregnant rats. In contrast, cervical desmin protein levels increased approximately 10-fold at DP21 compared to those in ovariectomized rats, but decreased rapidly at DB1, indicating its decrease at parturition and an inconsistency between mRNA and protein expression. The administration of 17ß-estradiol to ovariectomized rats increased desmin protein levels in the myometrium and cervix after 24 h. S-nitrosylated desmin protein was detected in the myometrium and cervix at DP21. The mRNA expression of inducible nitric oxide synthase was consistent with the expression of desmin protein. Thus, desmin, which is regulated by estradiol, is differentially expressed in the myometrium and cervix at peripartum possibly for successful pregnancy and parturition. In the cervix, desmin protein expression seems to be regulated by estradiol at the translational level. S-nitrosylation of desmin may have a potential role in the peripartum uterus.
Assuntos
Colo do Útero , Miométrio , Gravidez , Feminino , Ratos , Animais , Miométrio/metabolismo , Desmina/metabolismo , Útero/metabolismo , Parto , Estradiol/metabolismo , RNA Mensageiro/genéticaRESUMO
mRNA expression of molecules related to the activity of nitric oxide or prostaglandin E2, the critical regulators maintaining the ductus arteriosus patency, was examined in rat ductus arteriosus at preterm (days 18.5 and 19.5 of pregnancy) and near term (days 20.5 and 21.5). The endothelial nitric oxide synthase mRNA level increased transiently at preterm and then decreased at near term. The cyclooxygenase 2 mRNA increased gradually from near-term to the term complementary to the reduced endothelial nitric oxide synthase mRNA. These results suggest that the role shift between nitric oxide and prostaglandin E2 in maintaining ductus arteriosus patency at preterm and near term may be due to complementary expression changes of endothelial nitric oxide synthase and cyclooxygenase 2 at the transcriptional level.
Assuntos
Permeabilidade do Canal Arterial , Canal Arterial , Gravidez , Feminino , Ratos , Animais , Óxido Nítrico/metabolismo , Canal Arterial/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Dinoprostona/metabolismo , Prostaglandinas/metabolismo , Ciclo-Oxigenase 2/metabolismo , RNA Mensageiro/metabolismoRESUMO
Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine ß-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca2+ entry and increased mRNA expression of voltage-gated Ca2+ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca2+ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.
Assuntos
Sulfeto de Hidrogênio , Tecido Adiposo/metabolismo , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Óxido Nítrico/metabolismo , RNA Mensageiro , Células-Tronco/metabolismo , EnxofreRESUMO
The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.
Assuntos
Tecido Adiposo/citologia , Canais de Cálcio Tipo N/metabolismo , Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/citologia , Ácido Valproico/farmacologia , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura , Masculino , Neurônios/efeitos dos fármacos , Ratos Wistar , Células-Tronco/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The involvement of nitric oxide (NO) signaling in apoptosis was examined in the placental bed of mid-to-late pregnant rats. Pregnant rats were treated with l-NAME, a nitric oxide synthase inhibitor, by subcutaneous infusion for 48 hours before the examination at day 13.5, 17.5, or 21.5. l-NAME induced apoptosis in the placental bed to a limited extent at days 13.5 and 17.5, but not at day 21.5. When the placental bed was examined at day 17.5, the protein expression of both executioner (C-Cas3) and inhibitor (XIAP) of apoptosis was increased by l-NAME, but they did not co-localized with apoptosis. It was presumed that placental bed apoptosis induced by l-NAME is regulated through the expression of both executioner and inhibitor, possibly involving protein S-nitrosylation.
Assuntos
NG-Nitroarginina Metil Éster , Placenta , Animais , Apoptose , Inibidores Enzimáticos , Feminino , Proteínas Inibidoras de Apoptose , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Gravidez , RatosRESUMO
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citrulina/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Ácido Valproico/farmacologia , Tecido Adiposo/citologia , Animais , Arginina/farmacologia , Argininossuccinato Sintase/antagonistas & inibidores , Argininossuccinato Sintase/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , N-Metilaspartato/análogos & derivados , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
Sika deer (Cervus nippon) in Japan are classified into southern and northern groups. However, previous studies primarily relied on maternally inherited mitochondrial DNA (mtDNA). The paternally inherited Y-chromosome is useful for analyzing the contribution of males to the population genetic history of sika deer. In total, approximately 16 kb of partial sequences of four Y-chromosomal genes, Y-linked, sex-determining region Y, DEAD-box helicase 3 Y-linked, and Zinc finger protein Y-linked, were sequenced to investigate intraspecific variation. As a result, we identified nine intronic single nucleotide polymorphisms (SNPs) in 478 sika deer samples collected over the entire Japanese archipelago from Hokkaido to Kyushu. SNP genotyping revealed 10 distinct haplotypes (SYH1-SYH10). The most common haplotype (SYH1) was present in all populations and was the most abundant haplotype, identified in 80.3% of the sampled individuals. The remaining haplotypes were unique to a single locality. SYH1 was also central to all other haplotypes that diverged by a SNP, resulting in this haplotype being the core of a star-like cluster topography. We found that contrary to mtDNA patterns, there was no clear differentiation of Y-chromosome markers between the southern and the northern populations. Due to the female philopatry of sika deer, mtDNA may provide a highly structured differentiation of populations. On the other hand, the male-biased gene flow may provide a reduced differentiation of populations. Our findings revealed that the genetic structure of the Japanese sika deer is more complex than previously thought based on mtDNA-based phylogeographic studies.
Assuntos
Cervos/genética , Cromossomo Y/genética , Animais , Genótipo , Japão , Masculino , Filogeografia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Interleukin-4 (IL4) and interleukin-13 (IL13) are involved in the initial response of T helper 2 lymphocytes through the activation of the IL4 receptor alpha (IL4RA), which is a common receptor chain for these cytokines. In humans, several single-nucleotide polymorphisms (SNPs) identified in the IL4R and in interleukin coding genes were associated with atopic disorders. However, the association between canine IL4R polymorphisms and atopic disorders has not been investigated yet. This study aimed to determine the associations between four non-synonymous SNPs and canine atopic dermatitis (CAD) in shiba inu and miniature dachshund populations. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to genotype four polymorphisms of canine IL4R and IL13 in 34 shiba inu and 19 miniature dachshund patients with CAD, as well as 29 shiba inu and 39 miniature dachshund patients without the condition. Results from miniature dachshunds revealed a potential association between the presence of minor A allele rs24378020 and CAD (odds ratio, 0.10; 95% confidence interval, 0.01-0.85; Poriginal=0.0062). This CAD resistance allele led to an amino acid substitution (Arg688Cys) that could impair IL4 and IL13 signaling. In shiba inu patients, rs24378020 was fixed by homozygosity of the major G allele. No association was found between the remaining three evaluated SNPs and CAD. Nevertheless, the study suggests that the IL4R Cys688 variant reduces the risk of CAD in miniature dachshunds.
Assuntos
Dermatite Atópica , Doenças do Cão , Interleucina-13 , Receptores de Interleucina-4 , Animais , Dermatite Atópica/genética , Dermatite Atópica/veterinária , Doenças do Cão/genética , Cães , Predisposição Genética para Doença , Genótipo , Interleucina-13/genética , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-4/genéticaRESUMO
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing ßIII-tubulin protein, a neuronal marker, by treatments with VPA at 2â¯mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2â¯h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400â¯W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as ßIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Ácido Valproico/farmacologia , Tecido Adiposo/citologia , Animais , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Guanilil Ciclase Solúvel/metabolismoRESUMO
Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679â¯hPa) or positive (1347â¯hPa) air pressure applied with a disposable syringe, and shaking (60â¯rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140-170â¯mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.
Assuntos
Pressão do Ar , Permeabilidade da Membrana Celular/fisiologia , Crioprotetores/metabolismo , Dimetil Sulfóxido/metabolismo , Etilenoglicol/metabolismo , Ovário/efeitos dos fármacos , Animais , Bovinos , Contagem de Células , Criopreservação/métodos , Feminino , Ovário/citologia , VitrificaçãoRESUMO
Phase-contrast microscopy is a convenient technique for live-cell observation on the surface of materials with high optical transmittance. Here, we demonstrate a novel technique to observe living cells on the surface of materials with low optical transmittance, such as polytetrafluoroethylene (PTFE), which are widely used in biomaterials for blood-contacting devices. The surface of a cover glass was coated with a thin PTFE layer with sufficient transmittance, thereby enabling the observation of living cells on the PTFE surface with a phase-contrast microscope.
RESUMO
The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13-cis-retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.
Assuntos
Movimento Celular/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/toxicidade , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Técnicas In Vitro , Masculino , RatosRESUMO
Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker ßIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neurônios/citologia , Células-Tronco/citologia , Ácido Valproico/farmacologia , Acetilação/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Células Cultivadas , Expressão Gênica , Histonas/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , Ratos Wistar , Células-Tronco/metabolismo , Estimulação Química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Hepatoprotective effects of Rhizopus oryzae/â U-1 aqueous extract (RU) were demonstrated in carbon tetrachloride (CCl4 )-induced liver-injured rats. In order to investigate the RU effects, the rats were administered RU at a dose of 10 or 100 mg/kg of body weight for 10 days before induction of the liver injury by oral administration of CCl4 (125 mg/kg body weight). (i) Pretreatment with RU caused a significant decrease in serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities that were increased by the administration of CCl4 . (ii) RU pretreatment (100 mg/kg) increased 5-bromo-2'-deoxyuridine incorporation at 48 h after CCl4 treatment in hepatocytes. (iii) Histological hematoxylin and eosin staining of the liver showed that RU pretreatment reduced the damage induced by CCl4 administration. (iv) Reverse transcriptase PCR analysis showed RU retreatment caused a transient but significant increase in hepatocyte growth factor (HGF) and a sustained and significant increase in insulin-like growth factor-I (IGF-I) gene expression in hepatocytes injured by CCl4 treatment. From these results, we conclude that oral pre-administration of RU was effective to suppress liver injury induced by the subsequent oral CCl4 administration, and RU-induced increase in IGF-I and HGF gene expression may be, even in part, involved in biological actions of RU in rats.
Assuntos
Tetracloreto de Carbono/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fitoterapia , Extratos Vegetais/administração & dosagem , Rhizopus , Administração Oral , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bromodesoxiuridina/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , L-Lactato Desidrogenase/sangue , Masculino , Extratos Vegetais/farmacologia , Ratos Wistar , ÁguaRESUMO
Bifidobacterial plasmids reported so far are derived from a limited number of strains and plasmids of bifidobacterial type strains isolated from humans are unknown. We found that Bifidobacterium kashiwanohenseâ JCM 15439 (type strain) isolated from a healthy infant contained two cryptic plasmids, designated pBBKW-1 and pBBKW-2. We determined and analyzed the complete sequences of both plasmids. pBBKW-1 (7716 bp) was predicted to replicate by a rolling-circle mechanism and encode six protein-coding genes, two of which are putative replication proteins. pBBKW-1 seems to be a cointegrate plasmid containing two copies of the plasmid pMG1 from Bifidobacterium longum. pBBKW-2 (2920 bp) was predicted to encode six protein-coding genes and be a theta-type replicating plasmid, which has been reported to be more stable than a rolling circle-type replicating plasmid frequently found in bifidobacteria. Our finding will provide new insights into safe recombinant plasmid constructions for humans.
Assuntos
Sequência de Bases , Bifidobacterium/genética , Fezes/microbiologia , Plasmídeos/genética , Análise de Sequência de DNA , Bifidobacterium/isolamento & purificação , Humanos , Lactente , Dados de Sequência MolecularRESUMO
Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.
Assuntos
Tecido Adiposo/citologia , Proliferação de Células/efeitos dos fármacos , Cães/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco/fisiologia , Ácido Valproico/farmacologia , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Relação Dose-Resposta a Droga , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/fisiologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Células-Tronco/citologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/fisiologia , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/fisiologiaRESUMO
A polymorphic tetranucleotide (GAAT)n microsatellite in the first intron of the canine tumor necrosis factor alpha (TNFA) gene was characterized in this study; 139 dogs were analyzed: 22 Beagles, 26 Chihuahuas, 20 Miniature Dachshunds, 24 Miniature Poodles, 22 Pembroke Welsh Corgis and 25 Shiba Inus. We detected the presence of the 4 alleles (GAAT)5, (GAAT)6, (GAAT)7 and (GAAT)8, including 9 of the 10 expected genotypes. The expected heterozygosity (He) and the polymorphic information content (PIC) value of this microsatellite locus varied from 0.389 to 0.749 and from 0.333 to 0.682, respectively, among the 6 breeds. The allelic frequency differed greatly among breeds, but this microsatellite marker was highly polymorphic and could be a useful marker for the canine TNFA gene.
Assuntos
Cães/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , DNA/química , DNA/genética , Íntrons/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Óxido Nítrico/biossíntese , Rhizopus/química , Água/química , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo II/genética , Pirrolidinas/farmacologia , Ratos , Superóxidos/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.