Evaluación de la obturación de dos sistemas de cono único vs condensación lateral / Evaluation of two systems shutter single cone vs lateral condensation

El objetivo de este estudio fue evaluar in vitro la calidad de la obturación de 2 sistemas de obturación utilizando cono único (ProTaper y Mtwo) y la condensación lateral. Se usó 68 conductos vestibulares en 34 molares superiores, los cuales se separaron en 4 grupos de 15 muestras cada uno, dejando 8 raíces como control positivo y negativo. El Grupo A y C se instrumentó con Mtwo, Grupo B y D se instrumentó con ProTaper . Se obturaron los grupo A y B con condensación lateral, el grupo C con cono único Mtwo y el grupo D con cono único ProTaper. Luego de obturadas, se impermeabilizaron con esmalte de uñas y se colocaron en tinta china por 72 horas. Pasado este periodo, se retiró el esmalte de uñas y se procedió a diafanizar todas las muestras. Los dientes fueron fotografiados en papel milimetrado y medidos mediante el software Image Tools v.3.00 para medir en milímetros la tinta que se infiltró en la raíz por el tercio apical. Los resultados mostraron que no hubo diferencia estadísticamente significantes entre las técnicas de obturación utilizadas

The aim of this study was to compare in vitro the apical seal of single cone (ProTaper and Mtwo) and lateral condensation. 68 buccal canals were used in 34 upper molars, 4 groups of 15 samples each were formed, 8 roots used as positive and negative control. Group A and C were prepared with Mtwo, Group B and D were instrumented with ProTaper. The roots Were filled in groups A and B with lateral condensation, the single cone were used in group C with Mtwo and group D single cone with ProTaper. After sealed, waterproofed it with nail polish and placed in India ink for 72 hours. Then, withdrew the nail polish and proceeded to diaphanized all samples. The teeth were photographed and measured on graph paper using the Image Tools software v.3.00 for measuring in millimeters the ink infiltrated in the root apical third. results showed no statistically significant difference between filling techniques used

Cyclooxygenase-2 inhibition restores ultraviolet B-induced downregulation of ATP2A2/SERCA2 in keratinocytes: possible therapeutic approach of cyclooxygenase-2 inhibition for treatment of Darier disease.

BACKGROUND: ATP2A2 encoding the sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase2 (SERCA2) is a Darier disease (DD)-related gene. Ultraviolet (UV) B irradiation downregulates ATP2A2/SERCA2 expression in keratinocytes, whereas cyclooxygenase-2 (COX-2) expression is dramatically upregulated by UVB. OBJECTIVES: To analyse the involvement of COX-2 in ATP2A2/SERCA2 expression. METHODS: Keratinocytes were transfected with COX-2 siRNA or treated with COX-2 inhibitor, celecoxib, to evaluate the effect of COX-2 on ATP2A2/SERCA2 expression. Quantitative real-time polymerase chain reaction, Western blotting analysis and reporter assay were used to determine the amount of mRNA, protein level and transcription activity, respectively. RESULTS: COX-2 knockdown by siRNA resulted in upregulation of ATP2A2 transcription. Treatment by celecoxib rescued UVB-mediated suppression of the ATP2A2 transcription and SERCA2 protein expression. Simple addition of prostaglandin (PG) E(2) , which is a product of COX-2 enzyme, reduced the amounts of ATP2A2 mRNA and SERCA2 protein in keratinocytes. CONCLUSIONS: UVB downregulates ATP2A2/SERCA2 expression via induction of COX-2 expression and subsequent increase of PGE(2) production in keratinocytes. Considering that DD is caused by the decreased function of SERCA2 due to the reduced expression of the ATP2A2 gene, this finding shows the possibility that COX-2 inhibition may be useful to prevent and/or treat DD.

An association of TLR2­16934A >T polymorphism and severity/phenotype of atopic dermatitis.

BACKGROUND: Toll-like receptor 2 gene (TLR2) ­16934A>T polymorphism has been shown to be associated with severity of atopic dermatitis (AD) as measured using severity scoring of atopic dermatitis (SCORAD) index. Moreover, TLR2­16934A>T polymorphism has been associated with atopy and allergic disorders in farmers' children. OBJECTIVE: The aim of this study was to evaluate an association between TLR2­16934A>T polymorphism and AD phenotype, including disease severity and concomitant atopic diseases, or potential serum markers of AD severity and also to find a molecular background of the clinical associations. METHODS: Genotyping for TLR2­16934A>T polymorphism was performed in 130 consecutive adult ambulatory patients with AD. Total serum (TS) IgE levels, serum tryptase, plasma interleukin-6 and C-reactive protein were measured. In addition, luciferase assay and electrophoretic-mobility shift assay were conducted to assess the effect of ­16934A>T polymorphism on transcriptional activity. RESULTS: There was an inverse association of TLR2­16934TT genotype and/or ­16934T allele with SCORAD, but not with TS IgE, tryptase or inflammatory markers. Interestingly, ­16934AA genotype and/or ­16934A allele were overrepresented in AD patients with concomitant asthma or a family history of atopy. In a subgroup analysis, TLR2­16934A>T polymorphism was associated with SCORAD, asthma, allergic conjunctivitis or family history of atopy in AD patients with TS IgE ≥106 IU/mL but not in those having TS IgE <106 IU/mL. Functional analyses showed that TLR2­16934T allele is associated with higher luciferase activity in human monocytic THP-1 cells and preferential binding of the THP-1-derived nuclear protein. CONCLUSION: TLR2­16934A>T polymorphism could be a genetic predictor of AD severity, the coexistence of asthma or atopic conjunctivitis as well as a family history of atopic diseases, especially in subjects having higher TS IgE. TLR2­16934A>T polymorphism affects transcriptional activity, which may at least in part account for the clinical associations observed for the ­16934A>T polymorphism.

FcepsilonRIalpha gene (FCER1A) promoter polymorphisms and total serum IgE levels in Japanese atopic dermatitis patients.

Two promoter polymorphisms of the high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) gene (FCER1A), -66T>C (rs2251746) and -315C>T (rs2427827), were analysed in Japanese atopic dermatitis subjects. Patients with the -315CT/TT genotype tended to have higher total serum IgE levels, while the proportion of -315CT/TT genotype or the -315T allele was significantly higher in those with highly elevated total serum IgE concentrations.

Interaction of functional FCER2 promoter polymorphism and phenotype-associated haplotypes.

Low-affinity IgE receptor gene (FCER2) rs3760687 polymorphism was found to be associated with differential binding affinity of transcription factors Sp1 and Sp3 leading to altered transcriptional activity. Haplotypic interaction of functional FCER2 polymorphisms (rs28364072, rs2228137 and rs3760687) might potentially provide a background for genotype-phenotype associations previously observed for some rather non-functional FCER2 variants.

Functional analysis of a polymorphism in the promoter region of the IL-12/23p40 gene.

BACKGROUND: Human IL-12B gene on chromosome 5q31 encodes the common p40 subunit of IL-12 and IL-23. IL-12 is known to play critical roles in the generation of T-helper type 1 (TH(1)) cells, whereas IL-23 is involved in maintenance and/or population expansion of TH(17) cells. Although several reports suggested an association between a polymorphism (-6415CTCTAA/GC) in IL-12B and asthma, the molecular mechanism how this polymorphism is involved in allergic inflammation is still unclear. METHODS: The transcription activity was analysed by reporter assay. A transcription factor binding to -6415 polymorphic site was identified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The amount of cytokines produced from peripheral monocytes were determined by ELISA. RESULTS: Reporter assay showed that the transcription activity of the GC allele was higher than that of the CTCTAA allele. A transcription factor Sp1 bound to the region including the GC allele with a higher affinity than that of the CTCTAA allele in EMSA. In vivo binding of Sp1 to IL-12B gene carrying -6415GC was confirmed by ChIP assay. Overexpression of Sp1 up-regulated transcription activity of promoter carrying GC allele sequence, whereas the CTCTAA promoter was not affected by Sp1. We examined the correlation between -6415CTCTA/GC polymorphism and production of cytokine IL-12/23p40, IL-12p70, and IL-23 on peripheral blood monocytes, and monocytes with the GC/GC allele exhibited significantly higher expression of IL-12p70 protein than those with the CTCTAA/CTCTAA allele (P=0.009). CONCLUSIONS: The -6415 polymorphism is involved in cytokine production potential by affecting Sp1-mediated transcription activity.

FCER1A gene proximal promoter polymorphisms in Caucasians and East Asians.

Two FCER1A gene proximal promoter polymorphisms, -344C > T and -95T > C, both associated with total serum IgE and/or allergic disorders in Caucasians and East Asians, were shown to influence the gene expression in additive manner. In face of the rarity of other proximal promoter variants in Caucasians or their lack in East Asians, future investigations of the FCER1A locus in these ethnic groups should probably focus on three common haplotypes of the common -344C > T and -95T > C polymorphisms.

In vitro antimicrobial activity of Fill Canal, Sealapex, Mineral Trioxide Aggregate, Portland cement and EndoRez.

AIM: To determine in vitro the antimicrobial activity of Fill Canal, Sealapex, Mineral Trioxide Aggregate (MTA), Portland cement and EndoRez on various species of microorganisms. METHODOLOGY: The diffusion method on Müller-Hinton agar (MH) was employed. A base layer was made using MH agar and five wells were made by removing agar at equidistant points. Sealers were placed into the wells immediately after manipulation. The microorganisms Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were seeded by pour plate. The plates were kept at room temperature for 2 h for prediffusion and then incubated at 37 degrees C for 24 h. Aliquots of 10 mL of 0.05% triphenyltetrazolium chloride gel were added for optimization and the zones of inhibition were measured. RESULTS: Sealapex and Fill Canal demonstrated antimicrobial activity for all strains. For MTA and Portland cement, only E. coli was not inhibited. No antimicrobial activity was detected for EndoRez. CONCLUSIONS: In this laboratory study, Fill Canal, Sealapex, MTA and Portland cement presented antimicrobial activity whilst EndoRez did not.

Cloning of full-length genomic DNA encoding human FcepsilonRI alpha-chain and its transcriptional regulation.

Two novel exons, named exon 1A and exon 2A, were found at 18.4 and 12.6 kb upstream from the exon known as the first exon of human FcepsilonRI alpha-chain gene. Transcription from the promoter present in the upstream of exon 1A was decreased by mutations introduced into the "first intron" between exon 1A and exon 2A, suggesting the presence of an intronic regulatory element in the intron. Consistent with this, electrophoretic mobility shift assay revealed the presence of a nuclear factor which bound the region in FcepsilonRI alpha-chain positive cells.

[A case of Folliculitis barbae Candidomycetica].

A 71-year-old man was referred to our department on January 30, 1998 with hard red papules that had developed on the philtrum in mid-January. On January 2, the patient had received high-dose steroid therapy (pulse therapy) for cluster asthma attacks and antibiotics at the Department of Internal Medicine of our hospital. Infiltrative, protruding reddish plaques were observed on the philtrum, which contained a number of small pustules at sites corresponding to hair follicles. There was partial opacity and slight irregularity of the nail plates on the first and second toes of the right foot. Fungal elements were detected from a lesion on the mustache and the nail. Histological examination of the lesion on the philtrum revealed infiltration of inflammatory cells comprising neutrophils, lymphocytes, and macrophages around the hair follicles. Beard hair and nail cultures revealed Candida albicans A, indicating that the patient had candidal sycosis and candidal onychia. He was treated with oral fluconazole (100 mg/day). The lesion was clinically improved within 50-days. Recently, extensive use of steroids and antibiotics has produced an increase in reports of patients with Folliculitis barbae Candidomycetica. We believe that the present case was also induced by high-dose steroid therapy and antibiotics.

Molecular cloning of rat USF2 cDNA and characterization of splicing variants.

The complete nucleotide sequence of rat USF2 cDNA was determined. In addition to the full length clone (USF2FL), four isoforms (delta1, delta2, delta3, and delta4) suggested to be generated by alternative splicing were isolated. USF2delta1 and delta2 lacked 27 and 67 internal amino acid residues, respectively. USF2delta3 and delta4 lacked most of the entire sequence but encoded short peptides of an N-terminal portion of USF2FL. Overexpression of USF2FL increased the transcription of the human high affinity IgE receptor (FcepsilonRI) alpha chain gene through specific binding to the CAGCTG motif in the first intron. On the other hand, overexpression of USF2delta1 or delta2 reduced the transcription of the human FcepsilonRI alpha chain gene. Both USF2FL and USF2delta1 bound to CACGTG as well as CAGCTG, while USF2delta2 bound to CACGTG but not to CAGCTG. These results suggested the presence of a different and definitive role of each variant in the expression of the alpha chain gene.

A complex composed of USF1 and USF2 activates the human FcepsilonRI alpha chain expression via a CAGCTG element in the first intron.

The high-affinity IgE receptor, FcepsilonRI, is a key regulatory molecule in the allergic reaction. During the course of studies to find cis-acting elements for FcepsilonRI alpha chain gene expression, a CAGCTG sequence located in the first intron was revealed to serve as a crucial enhancer element. Electromobility shift assays using antibodies and in vitro translation products showed that the CAGCTG element was recognized by the USF1/USF2 complex. As was the case for other intronic cis-elements, the CAGCTG element regulated the promoter in an orientation- and position-dependent manner. Overexpression of USF2 antisense repressed the FcepsilonRI alpha chain gene promoter and decreased the amount of alpha chain mRNA in mast cell lines. All these results indicated that the USF1/USF2 complex activates the human FcepsilonRI alpha chain gene expression via the CAGCTG element in the first intron.

Splice isoforms of transcription factor Elf-1 affecting its regulatory function in transcription-molecular cloning of rat Elf-1.

To elucidate the role of Elf-1 in Fc epsilonRI alpha chain expression, rat Elf-1 cDNAs were isolated and characterized. The rat Elf-1 cDNA of 2744 bp contained an open reading frame of 1848 bp. In addition to the full length rat Elf-1 cDNA (named type 1), two splice isoforms were isolated. One of the two isoforms lacked the amino acid residues from 85th to 120th (type 2), and the other from 85th to 175th (type 3). Similar isoforms were also observed in human tissue. Overexpression of rat Elf-1 (type 1) using a transient coexpression system inhibited of the alpha chain promoter activity. The inhibition activity was different between the isoforms; the inhibition activity of type 2 was lower than that of type 1, and type 3 did not have an inhibitory effect. This observation suggested that each Elf-1 isoform played a different role in the gene expression under its control.

The transcription factors Elf-1 and GATA-1 bind to cell-specific enhancer elements of human high-affinity IgE receptor alpha-chain gene.

Key regulatory regions necessary for the expression of the gene encoding FcepsilonRI alpha-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, -74/-69 and -55/-47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the alpha-chain promoter. Both the regulatory elements enhanced the promoter activity only in alpha-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the alpha-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcepsilonRI alpha-chain.

Analysis of human IgE epitope of Der f 2 with anti-Der f 2 mouse monoclonal antibodies.

Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.

Dehydration modifies somal CRH immunoreactivity in the rat hypothalamus: an immunocytochemical study in the absence of colchicine.

Corticotropin-releasing hormone immunoreactive (CRH-ir) neurons were examined in the hypothalamus of euhydrated and dehydrated rats without using colchicine. CRH-ir cells were observed in the lateral hypothalamus, retrochiasmatic, and in magnocellular parts of the supraoptic and paraventricular hypothalamic nucleus (PVH) in dehydrated but not euhydrated animals. However, CRH-ir neurons were decreased in the medial parvicellular part of the PVH. These results indicate that altered CRH mRNA levels previously reported in dehydrated animals translate into changes in peptide immunoreactivity.

Functional interactions between nuclear receptors recognizing a common sequence element, the direct repeat motif spaced by one nucleotide (DR-1).

Direct repeat motifs composed of two hexamer half-sites spaced by a single nucleotide (DR-1) are recognized by several members of the nuclear hormone receptor superfamily. We examined, by means of gene transfection assays, the interplay between the DR-1-binding nuclear receptors commonly expressed in liver, peroxisome proliferator-activated receptor alpha (PPARalpha), hepatocyte nuclear factor-4 (HNF-4), and chicken ovalbumin upstream transcription factor I (COUP-TFI). Both PPARalpha and HNF-4 efficiently bound to the acyl-CoA oxidase gene enhancer element, but PPARalpha exhibited much stronger transactivation than HNF-4. As a result, HNF-4 suppressed the gene-activating function of PPARalpha, when they were expressed together, due to competition for a common binding site. On the other hand, HNF-4, but not PPARalpha, effectively bound to the apolipoprotein CIII gene element, and activated gene transcription. PPARalpha had no effect even when co-expressed with HNF-4. COUP-TFI bound to both elements, and suppressed the gene activation by PPARalpha and HNF-4. Thus, these nuclear receptors have individual functions in gene regulation, and exhibit complex compound effects when they co-exist.