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1.
Braz. oral res. (Online) ; 33: e058, 2019. tab, graf
Article in English | LILACS | ID: biblio-1019608

ABSTRACT

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Young Adult , Genetic Markers/genetics , Cell Culture Techniques/methods , Dental Cementum/cytology , Phosphates/pharmacology , Phosphoproteins/analysis , Phosphoproteins/genetics , Sialoglycoproteins/analysis , Sialoglycoproteins/genetics , Time Factors , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Gene Expression , Cell Line , Analysis of Variance , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Fluorescent Antibody Technique , Bone Morphogenetic Proteins/analysis , Bone Morphogenetic Proteins/genetics , Dental Cementum/metabolism , Adaptor Proteins, Signal Transducing , Molar/cytology
2.
Braz. j. microbiol ; 48(3): 566-569, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-889146

ABSTRACT

Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.


Subject(s)
Animals , Phosphoproteins/genetics , Rabies/veterinary , Rabies virus/genetics , Viral Proteins/genetics , Chiroptera/virology , RNA, Small Interfering/genetics , RNA Interference , Phosphoproteins/metabolism , Rabies/virology , Rabies virus/physiology , Viral Proteins/metabolism , Virus Replication , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism
4.
Article in English | WPRIM | ID: wpr-34568

ABSTRACT

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.


Subject(s)
Antiviral Agents/therapeutic use , Cytomegalovirus/genetics , Cytomegalovirus Infections/drug therapy , DNA, Viral/blood , Ganciclovir/therapeutic use , Humans , Immunoassay , Organ Transplantation , Phosphoproteins/genetics , Real-Time Polymerase Chain Reaction , Viral Matrix Proteins/genetics , Virology/methods
5.
Article in English | WPRIM | ID: wpr-64361

ABSTRACT

BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.


Subject(s)
Adolescent , Carrier Proteins/genetics , Child , Child, Preschool , Cohort Studies , Cytogenetic Analysis , DNA Mutational Analysis , Female , Gene Frequency , Genotype , Humans , Infant , Leukemia, Myeloid, Acute/genetics , Male , Nuclear Proteins/genetics , Phosphoproteins/genetics , Polymorphism, Single Nucleotide , RNA Splicing , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoproteins/genetics
6.
Braz. j. microbiol ; 45(3): 851-855, July-Sept. 2014. tab
Article in English | LILACS | ID: lil-727013

ABSTRACT

Enteropathogenic Escherichia coli (EPEC) are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.


Subject(s)
Humans , Bacterial Secretion Systems/genetics , Enteropathogenic Escherichia coli/genetics , Escherichia coli Proteins/genetics , Genetic Variation , Phosphoproteins/genetics , Virulence Factors/genetics , Enteropathogenic Escherichia coli/classification , Enteropathogenic Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Gastroenteritis/microbiology
7.
Article in English | WPRIM | ID: wpr-113786

ABSTRACT

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-alpha induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-alpha- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Apoptosis , Cell Line , Epithelial Cells/cytology , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins/genetics , Lysophospholipids/metabolism , Phosphoproteins/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , RNA, Small Interfering/genetics , Receptors, Lysophosphatidic Acid/genetics , Salivary Glands/cytology , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism
8.
Medicina (B.Aires) ; 73(4): 297-302, jul.-ago. 2013.
Article in Spanish | LILACS, BINACIS | ID: biblio-1165166

ABSTRACT

StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2A>G STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient’s testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13


of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0


± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2A>G) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.


Subject(s)
Phosphoproteins/genetics , Adrenal Hyperplasia, Congenital/genetics , Mutation/genetics , /genetics , Animals , Chlorocebus aethiops , COS Cells , Phenotype , Humans , Adrenal Insufficiency/genetics , Pedigree , Male , Polymorphism, Genetic , Real-Time Polymerase Chain Reaction , Infant, Newborn
9.
Braz. j. med. biol. res ; 46(8): 700-707, ago. 2013. tab, graf
Article in English | LILACS | ID: lil-684532

ABSTRACT

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.


Subject(s)
Animals , Cattle , Female , Culture Media/pharmacology , Estradiol/pharmacology , Ovarian Follicle/drug effects , Progesterone/pharmacology , Tissue Culture Techniques , Analysis of Variance , Aromatase/genetics , Culture Media, Serum-Free , Cholesterol Side-Chain Cleavage Enzyme/genetics , Gene Expression , Ovarian Follicle/anatomy & histology , Phosphoproteins/genetics , Progesterone Reductase/genetics , Reverse Transcriptase Polymerase Chain Reaction , Receptors, FSH/genetics , /genetics
10.
Indian J Exp Biol ; 2013 Feb; 51(2): 116-123
Article in English | IMSEAR | ID: sea-147574

ABSTRACT

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.


Subject(s)
Amino Acid Sequence , Animals , Annexin A5/metabolism , Base Sequence , Chickens , Cloning, Molecular , Gene Expression Regulation, Viral , Genes, Viral/genetics , HeLa Cells , Humans , Molecular Sequence Data , Newcastle disease virus/genetics , Open Reading Frames/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Reproducibility of Results , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
11.
Biol. Res ; 46(2): 139-146, 2013. ilus, tab
Article in English | LILACS | ID: lil-683990

ABSTRACT

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.


Subject(s)
Animals , Female , Male , Pregnancy , Diethylhexyl Phthalate/toxicity , Endocrine Disruptors/toxicity , Genistein/toxicity , Genitalia, Male/drug effects , Lactation/drug effects , Phytoestrogens/toxicity , Plasticizers/toxicity , Cytochrome P-450 CYP11B2/genetics , Maternal Exposure/adverse effects , Phosphoproteins/genetics , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class B/genetics , /genetics , Testis/drug effects
12.
Article in English | WPRIM | ID: wpr-148462

ABSTRACT

Congenital adrenal insufficiency is caused by specific genetic mutations. Early suspicion and definite diagnosis are crucial because the disease can precipitate a life-threatening hypovolemic shock without prompt treatment. This study was designed to understand the clinical manifestations including growth patterns and to find the usefulness of ACTH stimulation test. Sixteen patients with confirmed genotyping were subdivided into three groups according to the genetic study results: congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH, n=11), congenital lipoid adrenal hyperplasia (n=3) and X-linked adrenal hypoplasia congenita (n=2). Bone age advancement was prominent in patients with CAH especially after 60 months of chronologic age (n=6, 67%). They were diagnosed in older ages in group with bone age advancement (P<0.05). Comorbid conditions such as obesity, mental retardation, and central precocious puberty were also prominent in this group. In conclusion, this study showed the importance of understanding the clinical symptoms as well as genetic analysis for early diagnosis and management of congenital adrenal insufficiency. ACTH stimulation test played an important role to support the diagnosis and serum 17-hydroxyprogesterone levels were significantly elevated in all of the CAH patients. The test will be important for monitoring growth and puberty during follow up of patients with congenital adrenal insufficiency.


Subject(s)
17-alpha-Hydroxyprogesterone/blood , Disorder of Sex Development, 46,XY/drug therapy , Adolescent , Adrenal Hyperplasia, Congenital/drug therapy , Adrenal Insufficiency/congenital , Adrenocorticotropic Hormone/metabolism , Bone Development/genetics , Child , Child, Preschool , DAX-1 Orphan Nuclear Receptor/genetics , Female , Genetic Diseases, X-Linked/drug therapy , Genotype , Glucocorticoids/therapeutic use , Humans , Intellectual Disability/complications , Male , Mineralocorticoids/therapeutic use , Obesity/complications , Phosphoproteins/genetics , Puberty, Precocious/complications , Retrospective Studies , Steroid 21-Hydroxylase/genetics
13.
Article in English | WPRIM | ID: wpr-14962

ABSTRACT

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.


Subject(s)
3T3-L1 Cells , AMP-Activated Protein Kinases/metabolism , Adipocytes/drug effects , Adipogenesis/drug effects , Animals , Anti-Obesity Agents/pharmacology , Antigens, Differentiation/genetics , Carrier Proteins/genetics , Cell Size , Diet, High-Fat/adverse effects , Disease Models, Animal , Enzyme Activation , Gene Expression Regulation, Enzymologic/drug effects , Glycogen Synthase Kinase 3/genetics , Lipase/genetics , Lipolysis/drug effects , Male , Mice , Mice, Inbred C57BL , Obesity/etiology , Phosphoproteins/genetics , Phosphorylation , Propylene Glycols/pharmacology , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/metabolism , Sphingosine/analogs & derivatives , Sterol Esterase/metabolism
14.
Article in English | WPRIM | ID: wpr-164987

ABSTRACT

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of 'dendritic' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.


Subject(s)
Animals , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , Dendrites/metabolism , Hippocampus/cytology , Immunohistochemistry , In Situ Hybridization, Fluorescence , Phosphoproteins/genetics , Potassium Chloride/pharmacology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Rats , Rats, Sprague-Dawley , Up-Regulation/drug effects
15.
Braz. dent. j ; 20(5): 365-369, 2009. ilus
Article in English | LILACS | ID: lil-537541

ABSTRACT

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein that plays an important role in mineralized tissue formation by initiation of nucleation and modulation of mineral phase morphology. The purpose of the present study was to examine the immunoexpression of DMP1 in tooth germs of 7 human fetuses at different gestational ages (14, 16, 19, 20, 21, 23 and 24 weeks) comparing with completed tooth formation erupted teeth. The results showed the presence of DMP1 in the dental lamina, as well as in the cells of the external epithelium, stellate reticulum and stratum intermedium of the enamel organ. However, in the internal dental epithelium, cervical loop region and dental papilla some cells have not labeled for DMP1. In the crown stage, DMP1 was expressed in the ameloblast and odontoblast layer, as well as in the dentinal tubules of coronal dentin near the odontoblast area. Erupted teeth with complete tooth formation exhibited immunolabeling for DMP1 only in the dentinal tubules mainly close to the dental pulp. No staining was observed in the enamel, predentin or dental pulp matrix. DMP1 is present in all developing dental structures (dental lamina, enamel organ, dental papilla) presenting few immunoexpression variations, with no staining in mineralized enamel and dentin.


A proteína da matriz dentinária 1 (DMP1) é uma fosfoproteína ácida que tem sido relacionada diretamente ao processo de mineralização dos tecidos em formação sendo iniciadora do processo de nucleação e modulação da fase mineral. O objetivo desse trabalho foi avaliar a imunoexpressão da DMP1 em germes dentários em diferentes fases da odontogênese, obtidos de 7 fetos humanos em diversos estágios gestacionais (14, 16, 19, 20, 21, 23 e 24 semanas), comparando-se com dentes com rizogênese completa. Os resultados mostraram que a DMP1 esteve expressa na lâmina dentária, bem como, nas células do epitélio externo, retículo estrelado e estrato intermediário do órgão do esmalte. Diferentemente, no epitélio interno do órgão do esmalte, alça cervical e papila dentária algumas células não apresentaram a DMP1. Nas fases de coroa, os ameloblastos e odontoblastos apresentaram marcação positiva para a DMP1, bem como os túbulos dentinários da dentina coronária próximos à região odontoblástica. Os dentes com rizogênese completa exibiram marcação para a DMP1 apenas nos túbulos dentinários principalmente próximos à polpa dentária. Nenhuma marcação foi observada na matriz de esmalte ou pré-dentina, nem na polpa dentária. Concluímos que a DMP1 está presente em todas as fases da odontogênese, tanto na lâmina dentária, órgão do esmalte, bem como na papila dentária, com pequenas variações de nuances de expressão, estando ausente na dentina e esmalte mineralizados.


Subject(s)
Humans , Extracellular Matrix Proteins/biosynthesis , Phosphoproteins/biosynthesis , Tooth Germ/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix/metabolism , Fetal Development , Gene Expression , Immunohistochemistry , Odontoblasts/metabolism , Odontogenesis/physiology , Phosphoproteins/genetics
16.
Article in English | WPRIM | ID: wpr-71513

ABSTRACT

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Animals , Blastomeres/cytology , Cell Line , Embryonic Development/genetics , Feedback, Physiological , Frizzled Receptors/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Humans , Mutation , Phosphoproteins/genetics , Protein Binding , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Transfection , Wnt Proteins/genetics , Xenopus , Xenopus Proteins/genetics
17.
Arq. bras. med. vet. zootec ; 59(5): 1154-1162, out. 2007. ilus, tab
Article in Portuguese | LILACS | ID: lil-471196

ABSTRACT

Empregou-se a técnica de reação em cadeia pela polimerase precedida de transcrição reversa para detecção do vírus da cinomose canina (CC). Para a padronização da técnica foram selecionados quatro pares de oligonucleotídeos (P1, P2, N1, H1), baseados em seqüências dos genes da fosfoproteína, neuraminidase e hemaglutinina, sendo utilizadas três cepas vacinais de vírus da CC como controles positivos. Foram analisadas três amostras isoladas de cães com cinomose e quatro amostras provenientes de cães com suspeita clínica de cinomose. Não houve amplificação nas amostras com suspeita clínica da doença. Os resultados obtidos com os oligonucleotídeos P1 e N1 foram superiores aos de H1. Os oligonucleotídeos P2 foram considerados inapropriados para a detecção do vírus da CC. Os amplicons obtidos com os oligonucleotídeos P1, N1 e H1 foram clivados com endonucleases de restrição, sendo os perfis das amostras virais comparados aos da amostra vacinal Lederle, utilizada como referência. Um padrão similar de restrição foi observado em todas as amostras analisadas


The reverse transcription-polymerase chain reaction was used to detect canine distemper virus (CDV). Four oligonucleotide pairs were selected (P1, P2, N1, H1), based on the sequences of the phosphoprotein, hemagglutinin and nuraminidase genes for assay standardization, and three CDV vaccine strains were used as positive controls. Three viral isolates from dogs with canine distemper and four samples from animals clinically suspected of distemper were analysed. No amplification was detected in suspected samples. Results obtained by using P1 and N1 oligonucleotides were superior to those with H1 ones. P2 oligonucleotides were considered inadequate for CDV detection. Amplicons resulting from amplification of P1, N1 and H1 oligonucleotides were submitted to cleavage by restriction endonucleases and restriction patterns of viral samples were compared to that of Lederle strain used as reference. A similar restriction pattern was observed in all analysed samples


Subject(s)
Animals , Dogs/virology , Distemper/diagnosis , Polymorphism, Genetic , Reverse Transcriptase Polymerase Chain Reaction/methods , Phosphoproteins/genetics , Hemagglutinins/genetics , Neuraminidase/genetics
18.
Article in English | WPRIM | ID: wpr-634342

ABSTRACT

To investigate p120 catenin mRNA expression in Non-Hodgkin's lymphoma (NHL) cell lines (U937, Raji, Jurkat and Molt4) and normal lymphocytes and explore the relationship between p120 catenin and Non-Hodgkins lymphoma, total RNA sample was extracted by using TRIzol and reversely transcripted into cDNA. Polymerase chain reaction was performed to detect mRNA expression of p120 catenin in NHL cell lines U937, Raji, Jurkat and Molt4. Normal lymphocytes were used as control. It was found expressions of p120 catenin 1A and 3A mRNA were high in above-mentioned NHL cell lines, but neither p120 catenin 1A nor 3A was found in normal lymphocytes as shown by RT-PCR. It is concluded that both P120ctn1A and P120ctn3A mRNA transcripts were found in all NHL cell lines U937, Raji, Jurkat and Molt4 but they don't exist in normal lymphocytes, suggesting p120ctn possibly is of importance in diagnosis and therapy of lymphoma.


Subject(s)
Catenins/genetics , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Jurkat Cells , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/pathology , Phosphoproteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Biomarkers, Tumor/genetics , U937 Cells
19.
Article in English | WPRIM | ID: wpr-15697

ABSTRACT

We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolatioin and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.


Subject(s)
Sequence Homology, Amino Acid , Sequence Analysis, DNA , Rats , Protein Isoforms/genetics , Phosphorylation , Phosphoproteins/genetics , Molecular Sequence Data , Mice , Male , Humans , Gene Expression Profiling , Female , DNA, Complementary/chemistry , Cyclic AMP-Dependent Protein Kinases/physiology , Cloning, Molecular , Cell Line , Base Sequence , Animals , Amino Acid Sequence , Adult
20.
Medicina (B.Aires) ; 66(supl.2): 33-36, 2006.
Article in Spanish | LILACS | ID: lil-480136

ABSTRACT

Cattle are recognized as the major reservoir of STEC and the source of infection for human beings. Until recently, intervention strategies to decrease the contamination of meat products have been focused on the slaughter plant with the application of practices to reduce the contamination and proliferation of STEC. This has now changed following the development of intervention strategies in the farm. This could be one of the most important points of intervention to lower the incidence of human infection. Vaccines, probiotics, bacteriophages, and changes in production practices may be useful as strategies to control EHEC in the cattle. The application of such intervention measures could be difficult due to the fact that this zoonotic agent rarely causes disease in bovines. The HUS is endemic in Argentina, and the factors leading to this epidemiological situation remain unknown. However, intervention strategies undoubtedly will contribute to reduce the incidence of this zoonosis.


Subject(s)
Humans , Animals , Cattle , Disease Reservoirs/microbiology , Escherichia coli Infections/prevention & control , /pathogenicity , Bacterial Proteins , Bacteriophages/immunology , Escherichia coli Infections/immunology , /virology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/therapeutic use , Escherichia coli Vaccines/therapeutic use , Hemolytic-Uremic Syndrome/immunology , Hemolytic-Uremic Syndrome/prevention & control , Lactobacillus acidophilus , Phosphoproteins/genetics , Phosphoproteins/therapeutic use , Probiotics/therapeutic use , Shiga Toxin/biosynthesis , Shiga Toxin/immunology
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