RESUMEN
In mammalian cells, Smad2 and Smad3, two receptor-regulated Smad proteins, play crucial roles in the signal transmission of transforming growth factor-ß (TGF-ß) and are involved in various cell regulatory processes, including epithelial-mesenchymal transition-associated cell responses, that is, cell morphological changes, E-cadherin downregulation, stress fiber formation, and cell motility enhancement. Smad2 contains an additional exon encoding 30 amino acid residues compared with Smad3, leading to distinct Smad2 and Smad3 functional properties. Intriguingly, Smad2 also has an alternatively spliced isoform termed Smad2Δexon3 (also known as Smad2ß) lacking the additional exon and behaving similarly to Smad3. However, Smad2Δexon3 and Smad3 signaling properties have not yet been compared in detail. In this study, we reveal that Smad2Δexon3 rescues multiple TGF-ß-induced in vitro cellular responses that would become defective upon SMAD3 KO but does not rescue cell motility enhancement. Using Smad2Δexon3/Smad3 chimeric proteins, we identified that residues Arg-104 and Asn-210 in Smad3, which are not conserved in Smad2Δexon3, are key for TGF-ß-enhanced cell motility. Moreover, we discovered that Smad2Δexon3 fails to rescue the enhanced cell motility as it does not mediate TGF-ß signals to downregulate transcription of ARHGAP24, a GTPase-activating protein that targets Rac1. This study reports for the first time distinct signaling properties of Smad2Δexon3 and Smad3.
Asunto(s)
Movimiento Celular , Exones , Eliminación de Secuencia , Transducción de Señal , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta , Animales , Mamíferos/metabolismo , Proteína Smad2/química , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/deficiencia , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Exones/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
E-cadherin, an epithelial cell-specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus-dependent downregulation of mRNA encoding E-cadherin (CDH1 mRNA) in KRAS-mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K-Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E-cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS-mutated cancer cells acquire a novel mode of regulating E-cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.
Asunto(s)
Antígenos CD/genética , Cadherinas/genética , Regulación hacia Abajo/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Células A549 , Línea Celular Tumoral , Progresión de la Enfermedad , Molécula de Adhesión Celular Epitelial/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica/genética , Células Hep G2 , Humanos , Proteína Quinasa C/genética , ARN Mensajero/genética , Transducción de Señal/genéticaRESUMEN
Gonadal sex differentiation proceeds by the interplay of various genes including the transcription factors and secretory factors in a complex network. The sex-differentiating genes are expressed not only during early sex differentiation but also throughout the gonadal development and even in the adult gonads. In addition, the evidence that they actually function in the adult gonads have been accumulated from the studies using the conditional knockout mice. However, many previous studies were focused on one single gene though those genes function in a network. In this study, the expressions of various sex-differentiating genes were analyzed simultaneously in the adult testis of the Japanese quail (Coturnix japonica), whose testicular functions are dramatically changed by altering the photoperiod, to elucidate the roles of them in the adult gonad. Anti-Müllerian hormone (AMH) was significantly upregulated in the regressed testis induced by the short-day condition. The expressions of the transcription factors that promote AMH expression in mammals (SF1, SOX9, WT1 and GATA4) were also increased in the regressed testis. Moreover, AMH receptor (AMHR2) showed similar expression pattern to its ligand. We also analyzed the expressions of other transforming growth factor beta (TGFB) superfamily members and their receptors. The expressions of the ligands and receptors of TGFB family, and follistatin and betaglycan in addition to inhibin subunits were increased in the regressed testis. These results suggest that AMH is involved in the adult testicular functions of the Japanese quail together with other TGFB superfamily members.
RESUMEN
Zing finger AN1-type domain 3 (ZFAND3), also known as testis expressed sequence 27 (Tex27), is a gene found in the mouse testis, but its physiological function is unknown. We identified the full-length sequences of two isoforms (short and long) of ZFAND3 cDNA from Japanese quail and leopard gecko. This is the first cloning of avian and reptilian ZFAND3 cDNA. The two isoforms are generated by alternative polyadenylation in the 3'UTR and have the same ORF sequences encoding identical proteins. There were highly conserved regions in the 3'UTR of the long form near the polyadenylation sites from mammals to amphibians, suggesting that the features for determining the stability of mRNA or translation efficiency differ between isoforms. The deduced amino acid sequence of ZFAND3 has two putative zinc finger domains, an A20-like zinc finger domain at the N-terminal and an AN1-like zinc finger domain at the C-terminal. Sequence analysis revealed an additional exon in the genomic structures of the avian and reptilian ZFAND3 genes which is not present in mammals, amphibians, or fish, and this exon produces additional amino acid residues in the A20-like zinc finger domain. Expression analysis in Japanese quail revealed that the expression level of ZFAND3 mRNA was high in not only the testis but also the ovary, and ZFAND3 mRNA was expressed in both spermatides of the testis and oocytes of the ovary. While the short form mRNA was mainly expressed in the testis, the expression level of the long form mRNA was high in the ovary. These results suggest that ZFAND3 has physiological functions related to germ cell maturation and regulatory mechanisms that differ between the testis and ovary.
Asunto(s)
Coturnix/genética , ADN Complementario/química , Lagartos/genética , Ovario/metabolismo , Isoformas de Proteínas/metabolismo , Testículo/metabolismo , Animales , Femenino , Masculino , Datos de Secuencia Molecular , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. METHODS: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.
Asunto(s)
Quimiocinas/análisis , Liquen Plano Oral/inmunología , Receptores de Quimiocina/análisis , Quimiocinas/genética , Células Epiteliales/inmunología , Epitelio/inmunología , Humanos , Inmunidad Celular , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
This study seeks to assess the potential of a 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) as a transcutaneous vaccine against chronic periodontitis. Transcutaneous immunization (TCI) of mice with 40k-OMP alone elicited 40k-OMP-specific IgG antibody (Ab) responses in both serum and saliva. When administered with cholera toxin (CT) as adjuvant, TCI with 40k-OMP not only elevated IgG Abs as noted above, but also induced IgA responses in serum but not in saliva. Salivary IgG from mice given 40k-OMP alone or 40k-OMP plus CT showed higher binding levels to the 40k-OMP than did that of non-immunized mice. Ab-forming cell (AFC) analysis revealed high numbers of 40k-OMP-specific IgG AFCs in the spleen but low numbers in the salivary glands of mice given 40k-OMP alone or 40k-OMP plus CT. Since 40k-OMP-specific IgG inhibited the coaggregation of P. gingivalis vesicles and S. gordonii, TCI with 40k-OMP may be a useful tool in the quest to prevent P. gingivalis infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Bacteroidaceae/prevención & control , Periodontitis/prevención & control , Porphyromonas gingivalis/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Administración Cutánea , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Vacunas Bacterianas/administración & dosificación , Toxina del Cólera/administración & dosificación , Toxina del Cólera/farmacología , Inmunoglobulina A/sangre , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Peso Molecular , Saliva/inmunologíaRESUMEN
STATEMENT OF PROBLEM: The selective pressure technique has been recommended for making impressions of maxillary edentulous residual ridges. Although various methods for making impressions have been reported, a definitive procedure has not been clearly elucidated. PURPOSE: This in vitro study evaluated changes in impression pressure produced by different types of relief space and escape holes in the impression tray for making an impression of a simulated maxillary edentulous arch. MATERIAL AND METHODS: Silicone impression material (Exadenture) and a maxillary edentulous acrylic cast were used. A miniature pressure sensor was embedded at the mid-palatal suture (point-P) and at the left first molar area on the edentulous ridge (point-R). Three types of tray relief were used: no spacer (NS), a 0.36-mm-thick sheet of wax (SS), or a 1.40-mm-thick base plate wax (BS). Four types of escape holes were made: no hole (NH), or escape holes of 0.5, 1.0, or 2.0 mm in diameter (05H, 10H, and 20H, respectively) in the area opposing point-P. Twelve trays were formed using these relief space and escape hole combinations. The cast and tray were attached to a rheometer for applying a continuous isotonic force of 5.0 kgf and compressive speed of 120 mm/min. Impressions were made and measurement of pressure (kPa) began immediately prior to compression and continued until the materials had polymerized for 2 minutes, with a sampling time of 5 Hz. Measurements were performed 5 times for each tray. The data were analyzed using 3-way analysis of variance and the Bonferroni test (alpha=.05). RESULTS: At initial pressure, the data obtained at point-P showed significantly higher values for NSNH, NS05H, SSNH, and SS05H (range: 22.29 +/- 1.58 kPa to 29.96 +/- 1.41 kPa) than those at point-R (range: 18.61 +/- 1.12 kPa to 22.71 +/- 2.11 kPa). At end pressure, the data obtained from NSNH at point P showed a significantly higher value (25.36 +/- 1.69 kPa) than that of point-R (15.36 +/- 0.99 kPa) (P<.001), whereas data from NS10H and NS20H at point-P showed a significantly lower value (6.32 +/- 0.84 kPa and 4.50 +/- 0.42 kPa) than at point-R (15.50 +/- 0.49 kPa and 14.98 +/- 0.88 kPa) (P<.001). The data obtained from SS05H, SS10H, and NS20H at point-P showed significantly lower values (range: 3.72 +/- 0.44 kPa to 9.10 +/- 0.26 kPa) than those at point-R (range: 13.40 +/- 1.31 kPa to 14.40 +/- 0.98 kPa). Moreover, the data obtained from BSNH, BS05H, BS10H, and BS20H at point-P showed significantly lower values (range: 3.24 +/- 1.96 kPa to 10.20 +/- 1.84 kPa) than those of point-R (range: 11.69 +/- 1.01 kPa to 14.04 +/- 2.08 kPa). CONCLUSION: For making impressions of an edentulous maxilla, the data suggest that a tray with an escape hole 1.0 mm or larger or a spacer thickness of base plate wax (1.40 mm) be used.
Asunto(s)
Materiales de Impresión Dental/química , Técnica de Impresión Dental/instrumentación , Arcada Edéntula , Maxilar , Resinas Acrílicas , Análisis de Varianza , Arco Dental , Diseño de Equipo , Humanos , Modelos Dentales , Polímeros/química , Presión , Reología/instrumentación , Elastómeros de Silicona/química , Estrés Mecánico , Propiedades de Superficie , Transductores de Presión , Ceras/químicaRESUMEN
In this study, we have assessed the efficacy of the 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis as a nasal vaccine for the prevention of adult periodontitis. Mice nasally immunized with 40k-OMP and cholera toxin as mucosal adjuvant displayed significant levels of 40k-OMP-specific serum IgG1, IgG2b and IgA as well as mucosal IgA antibodies (Abs) in saliva and nasal secretions. Ab-forming cell (AFC) analysis confirmed the antibody titers by detecting high numbers of 40k-OMP-specific AFCs in spleen, salivary glands and nasal passages. Because 40k-OMP-specific IgG inhibited coaggregation of P. gingivalis vesicles and S. gordonii, it may be an important tool for the prevention of adult periodontitis.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/inmunología , Porphyromonas gingivalis/inmunología , Adyuvantes Inmunológicos , Administración Intranasal , Animales , Antígenos Bacterianos/inmunología , Inmunización , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Saliva/inmunología , Saliva/microbiologíaRESUMEN
The hemolysin from Prevotella intermedia was partially purified from culture supernatant and then characterized. The hemolysin produced a clear beta-hemolytic zone on a blood agar plate. Hemolytic activity was 2.5-fold greater in culture supernatant compared to that cell-associated. The isolation and purification procedure involved ammonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The activity of this hemolysin was stimulated by reductants such as cysteine, dithiothreitol, glutathione etc., and was lost upon oxidation. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Ca(2+), Mg(2+) and EDTA did not affect the activity. The optimal pH of this hemolysin was 7.5.
Asunto(s)
Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/aislamiento & purificación , Prevotella intermedia/metabolismo , Sustancias Reductoras/farmacología , Medios de Cultivo , Proteínas Hemolisinas/efectos de los fármacos , Calor , Concentración de Iones de Hidrógeno , Prevotella intermedia/crecimiento & desarrollo , Tripsina/metabolismoRESUMEN
The purpose of this study was to determine the usefulness of green tea catechin for the improvement of periodontal disease. The minimum inhibitory concentration (MIC) and bactericidal activity of green tea catechin against black-pigmented, Gram-negative anaerobic rods (BPR) were measured. Hydroxypropylcellulose strips containing green tea catechin as a slow release local delivery system were applied in pockets in patients once a week for 8 weeks. The clinical, enzymatic and microbiological effects of the catechin were determined. Green tea catechin showed a bactericidal effect against Porphyromonas gingivalis and Prevotella spp. in vitro with an MIC of 1.0 mg/ml. In the in vivo experiment, the pocket depth (PD) and the proportion of BPR were markedly decreased in the catechin group with mechanical treatment at week 8 compared with the baseline with significant difference. In contrast, PD and BPR were similar to the baseline and the value at the end of the experimental period in the placebo sites of scaled groups. The peptidase activities in the gingival fluid were maintained at lower levels during the experimental period in the test sites, while it reached 70% of that at baseline in the placebo sites. No morbidity was observed in the placebo and catechin groups without mechanical treatment. Green tea catechin showed a bactericidal effect against BPR and the combined use of mechanical treatment and the application of green tea catechin using a slow release local delivery system was effective in improving periodontal status.