Topical betamethasone butyrate propionate exacerbates pressure ulcers after cutaneous ischemia-reperfusion injury.

Ischaemia-reperfusion (I/R) is involved in the development of various organ diseases. There has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. However, there is no evidence-based treatment for early-stage PUs. Our objective was to assess the effects of topical steroid on the development of PUs after cutaneous I/R injury in mice. Cutaneous I/R was performed by trapping the dorsal skin between two magnetic plates for 12 h, followed by plate removal. Topical application of betamethasone butyrate propionate (BBP) in I/R areas significantly increased the size of PUs after I/R. The number of thromboses was increased, and CD31(+) vessels were decreased in the I/R area treated with topical BBP. The number of oxidative stress-associated DNA-damaged cells and apoptotic cells in the I/R area was increased by topical BBP treatment. In addition, the mRNA level of NADPH oxidase 4 (Nox4), the essential enzyme that produces reactive oxygen species, was significantly increased and that of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates the expression of antioxidant proteins, was inhibited in the I/R area treated by BBP. The number of CD68(+) macrophages and the level of transforming growth factor-beta in lesional skin were also decreased by BBP. These results suggest that a topical steroid might accelerate the formation of PUs induced by cutaneous I/R injury by aggravating oxidative stress-induced tissue damage. Topical steroids might not be recommended for the treatment of acute-phase decubitus ulcers.

Analysis of regulatory mechanisms of an insulin-inducible SHARP-2 gene by (S)-Equol.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between -4687 and -4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus.

Effect of acidic primers on adhesive bonding of tri-n-butylborane initiated adhesive resin to alumina.

The present study was conducted to evaluate the effect of acidic primers on adhesive bonding to sintered alumina. Alumina disk specimens were primed with one of the following acidic materials: Acryl Bond, All Bond II Primer B, Alloy Primer, Estenia Opaque Primer, Eye Sight Opaque Primer, M.L. Primer, MR. Bond, and Super-Bond Liquid. The disks were bonded with an adhesive resin (Super-Bond) initiated with a tri-n-butylborane (TBB) derivative, and bond strengths were determined. Average bond strength before thermocycling varied from 42.9 to 44.3 MPa, whereas post-thermocycling bond strength ranged from 22.0 to 42.8 MPa. Of the nine groups assessed, reduction of bond strength after thermocycling was not significant in three: Alloy Primer, Estenia Opaque Primer, and Eye Sight Opaque Primer. It can be concluded that phosphate-based primers are recommended for bonding sintered alumina with Super-Bond resin.

Effect of single-liquid acidic primers on bonding of a composite luting agent joined to a prefabricated alumina coping material.

PURPOSE: The aim of this study was to assess the effect of acidic primers on adhesive bonding to prefabricated alumina material designed for fixed restorations. METHODS: High-purity alumina disks (Procera AllCeram) were primed with one of the following materials: Acryl Bond, All Bond II Primer B, Alloy Primer, Estenia Opaque Primer, M.L. Primer, MR. Bond, and Super- Bond Liquid. The specimens were bonded with a dualpolymerizing luting composite (Variolink II). Unprimed specimen was prepared as the control. Bond strengths were determined both before and after thermocycling. RESULTS: Average bond strength before thermocycling ranged from 12.0 to 39.1 MPa, whereas average bond strength after thermocycling varied from 0.0 to 26.9 MPa. The statistically highest post-thermocycling bond strength was obtained with the use of the Alloy Primer, Estenia Opaque Primer, and M.L. Primer agents. CONCLUSION: It can be concluded that the use of either the Estenia or Alloy Primer material, which contain 10- methacryloyloxydecyl dihydrogen phosphate (MDP), or the M.L. Primer, which contains 6-methacryloyloxyhexyl phosphonoacetate (6-MHPA), is recommended for bonding the Procera alumina copings with the Variolink II composite.

Effect of single-liquid priming agents on adhesive bonding to aluminum oxide of a methacrylic resin.

The purpose of this study was to evaluate the effects of acidic primers on adhesive bonding to sintered aluminum oxide (alumina). Alumina disks were primed with one of the following materials: Acryl Bond, All Bond 2 Primer B, Alloy Primer, Estenia Opaque Primer, Eye Sight Opaque Primer, M.L. Primer, MR. Bond, and Super-Bond Liquid. Specimens were then bonded with an acrylic resin initiated with partially oxidized tri-n-butylborane (TBBO), and bond strengths were determined. Unprimed specimen was employed as the control. Average bond strength before thermocycling ranged from 20.5 to 41.9 MPa, whereas post-thermocycling bond strength ranged from 0.0 to 40.0 MPa. Of the eight primers, Estenia Opaque Primer and Alloy Primer showed better adhesive performance than the other materials. It could thus be concluded that either Estenia Opaque Primer or Alloy Primer--which contained an adhesive monomer, 10-methacryloyloxydecyl dihydrogen phosphate (MDP)--was recommended for bonding alumina with TBBO-initiated resin.

The mouse zinc-fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3.

Human zinc-fingers and homeoboxes (ZHX) 1, ZHX2 and ZHX3, members of the ZHX family, contain two Cys(2)-His(2)-type zinc-finger motifs and five homeodomains (HDs). These proteins not only form homodimers but heterodimers with ZHX1 as well and act as ubiquitous transcriptional repressors. The cloning of mouse ZHX2 and ZHX3 cDNAs and the corresponding genes from a 129 mouse genomic library are reported, along with an analysis of the heterodimerization of ZHX2 with ZHX3. The mouse ZHX2 and ZHX3 proteins consist of 836 and 951 amino acid residues, respectively. The similarity of amino acid sequences of each protein with those of human orthologue is 87.0% and 85.2%, respectively. An analysis of genomic clones revealed that an entire coding sequence and a portion of the 5'- and 3'-noncoding sequence of mouse ZHX2 cDNA are encoded by a single exon of the mouse ZHX2 gene as well as the mouse ZHX1 gene. In contrast, in the case of the mouse ZHX3 gene, the coding sequences of ZHX3 cDNA are separated by an intron. A 4.5-kb ZHX2 transcript, and three ZHX3 transcripts, 9.5-, 6.5- and 4.4-kb, are ubiquitously expressed, although their levels vary. Lastly, in vitro and in vivo protein-protein interaction assays revealed that ZHX2 is able to form a heterodimer with ZHX3 via a region containing each HD1.

Genomic structure and analysis of transcriptional regulation of the mouse zinc-fingers and homeoboxes 1 (ZHX1) gene.

The mouse zinc-fingers and homeoboxes 1 (ZHX1) gene was cloned and its transcriptional regulatory mechanism analysed. The mouse ZHX1 gene spans approximately 29 kb and consists of five exons. Exons 1-3 contain the nucleotide sequence of the 5'-noncoding region of mouse ZHX1 cDNA, exon 4 contains a part of the 5'-noncoding region, an entire coding sequence, and a part of the 3'-noncoding sequence, and exon 5 contains the resulting 3'-noncoding sequence. The ZHX1 gene exists as one copy in the haploid mouse genome. Two species of ZHX1 mRNA with or without the nucleotide sequence of the third exon are produced by an alternative splicing. To investigate the regulatory elements involved in the transcription of the ZHX1 gene, transient DNA transfection experiments with ZHX1/firefly luciferase reporter genes were performed using a lipofection method. Functional analyses of a series of 5'- and 3'-deletion constructs of the reporter genes revealed that the nucleotide sequence between -59 and +50 is required for full promoter activity in mouse embryonal carcinoma F9 cells. Two positive regulatory cis-acting elements in the region were identified. These elements, designated as Box A and Box B, are located between nucleotides -47 and -42 and +22 and +27, respectively, and synergistically stimulate transcription of the mouse ZHX1 gene. Electrophoretic mobility shift assays with specific competitors and antibodies show that PEA3 and Yin and Yang 1 (YY1) bind to Box A and Box B, respectively. Thus, we conclude that PEA3 and YY1 synergistically stimulate the transcription of the ZHX1 gene.

Rat zinc-fingers and homeoboxes 1 (ZHX1), a nuclear factor-YA-interacting nuclear protein, forms a homodimer.

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.

Early growth response gene-1 regulates the expression of the rat luteinizing hormone receptor gene.

LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.

Genetic identification of cinnamon (Cinnamomum spp.) based on the trnL-trnF chloroplast DNA.

Genetic identification among cinnamon species was studied by analyzing nucleotide sequences of chloroplast DNA from four species (Cinnamomum cassia, C. zeylanicum, C. burmannii and C. sieboldii). The two regions studied were the intergenic spacer region between the trnL 3'exon and trnF exon (trnL -trnF IGS) and the trnL intron region. We found nucleotide variation at one site in the trnL-trnF IGS, and at three sites in the trnL intron. With the sequence data from analysis of these regions, the four Cinnamomum species used in this study were correctly identified. Furthermore, single-strand conformation polymorphism (SSCP) analysis of PCR products from the trnL-trnF IGS and the trnL intron resulted in different SSCP band patterns among C. cassia, C. zeylanicum and C. burmannii.