Manufacturer's Directions Research Articles

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX, the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

Sealability of Filled and Unfilled Fissure Sealants using Two Different drying agents

To evaluate and compare the effect of two different drying agents (PrimaDry and Ethyl alcohol 95% concentration.) on microleakage of filled (Ultraseal XT plus) and unfilled (Helioseal) fissure sealants in vivo and in vitro. A total of 60 extracted human upper and lower first premolars from15 adult healthy patients were divided into 2 groups 30 in vivo (Teeth were extracted 3 months after sealant application), and 30 in vitro (Teeth were stored 3 months in normal saline) each group were subdivided into 2 subgroups 15 sealed with filled (Ultraseal XT plus), while the other 15 were sealed with unfilled (Helioseal), each subgroup was subdivided into 3 classes composed of (5) premolars according to drying agent used: B0) without drying agent. B1) PrimaDry B2) Ethyl alcohol 95% concentration. After extraction teeth were thoroughly washed with water, cleaned with non-fluoridated (FL) prophylaxis paste. The unsealed were sealed after 3 months of storage in saline solution (in vitro).The occlusal surfaces were opened using tapered diamond fissure burs confined to enamel. Fissure sealant application was done following the manufacturer's instructions. The drying agent was applied after etching and before sealant application. Dye penetration method with methylene blue dye was used for microleakage assessment. The specimens were immersed in 1% methylene blue for 24h in order to allow dye penetration into possible gaps in the tooth–sealant interface. The teeth were sectioned longitudinally in using diamond saw, each section cleaned, examined and photographed under stereomicroscope at ×20 magnifications to assess dye penetration at the margins of the restorations. Two-way ANOVA was used for statistical analysis. In vivo results revealed that unfilled (Helioseal) achieved significant less microleakage than filled (Ultraseal XT plus) without any drying agent and PrimaDry with either filled or unfilled achieved the least microleakage. While in vitro results showed that both PrimaDry with Filled (Ultraseal XT plus) and Ethyl alcohol 95% concentration with Unfilled (Helioseal) achieved the least microleakage without significant statistical difference. Use of Ultraseal XT plus system according to manufacturer's directions showed the least microleakage both in vivo and in vitro. Use of PrimaDry increased the efficiency of both filled and unfilled fissure sealant.

IV형 경석고와 경화처리된 IV형 경석고 그리고 폴리우레탄 수지 치형재의 표면경도와 미세구조 특성의 비교

Purpose: This study compared the surface hardness (Vickers) and microstructural characteristics between a type IV stone with and without die hardening treatment, a polyurethane die material. Methods: Materials used were a type IV stone(MG Crystal Rock), two die hardeners (Hardening bath, Epox-it), and a polyurethane resin material(Polyluck). Six specimens per group were prepared according to manufacturer's directions. The prepared specimens were tested by means of hardness test, one-way ANOVA analysis, scanning electron microscopic(SEM) observations and energy dispersive spectroscopic(EDS) analysis. Results: In the hardness test and its statistical analysis, there was no significant difference in the surface hardness between a type IV stone and type IV stone with die hardener coating, type IV stone mixed with an epoxy like material instead of water. In contrast, polyurethane resin material exhibited significantly greater surface hardness than other specimen groups (p<0.05). Conclusion: By considering the results of the hardness test, SEM observations and EDS analysis, although the die hardeners on type IV stone did not show remarkable improvement in surface hardness, the die hardener coating on the surface of type IV stone material did show decrease of microporous and improvement of surface defects.

ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases.

First Report of Hyphodermella rosae Causing Dry Fruit Rot Disease on Plum in Iran.

Plum (Prunus domestica) and peach (P. persica) are widely grown, often in alternate rows with citrus, in the Mazandaran Province of Iran. In June 2011, a dry fruit rot of plum was observed in several production regions in Mazandaran Province (35°47'N, 50°34'E). Initial symptoms at pit-hardening stage appeared as dark brown, circular, necrotic spots from 2 to 5 cm in diameter. They later developed into a dry fruit rot. Severe symptoms occurred during June and July when warm weather (temperature around 28°C) and high relative humidity (RH) (>85%) were present. Marketable yield losses reached 50% to almost 100% in many orchards. To isolate the causal organism, symptomatic fruits were surface disinfested for 1 min in 0.5% active chlorine, washed thoroughly with sterile distilled water, and segments were plated on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate and incubated at 25°C for 3 days. The fungus Hyphodermella rosae (Bresadola) Nakasone was consistently isolated (37 isolates from 79 samples) and identified on the basis of morphological characteristics on PDA. Basidiomata were effuse, resupinate, 15 × 10 mm, crustaceous, tubercules small with apical bristles, and light orange to grayish orange. Subhymenium was up to 30 μm thick, composed of vertically arranged, short-celled, nonagglutinated hyphae; subhymenial hyphae were 3 to 4 μm in diameter. Basidiospores were ellipsoid, 7.5 to 8.5 × 4.5 to 5.5 μm (100 determination), and their cell walls were thin, hyaline, and smooth (1). Genomic DNA was extracted from mycelium with a DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's directions and grown on potato dextrose broth for 4 days at 28°C. The rDNA region was amplified with the primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'- GGAAGTAAAAGTCGTAACAA-3') (4) and the PCR product was sequenced. Nucleotide BLAST analysis of the amplified 627-bp fragment confirmed a 99% similarity with the sequence of H. rosae (GenBank Accession No. JN593086). A pathogenicity test was conducted with isolate MA4099 by placing 5-day-old mycelial plugs grown on PDA at the surface of healthy fruit (n = 6) incubated under >85% RH at 25°C for at least 4 days until the appearance of symptoms, which were similar to those displayed under orchard conditions. Control fruits, inoculated with blocks of PDA plugs, remained intact and symptomless. Reisolation from inoculated fruit samples consistently yielded the inoculated fungus, completing Koch's postulates. The genus Hyphodermella has been reported to be causing wood rot on apricot (2) and sweet and sour cherry (3). To our knowledge, this is the first report of H. rosae causing dry fruit rot on a stone fruit species in the world.

Mon1a Protein Acts in Trafficking through the Secretory Apparatus

Mon1a was originally identified as a modifier gene of vesicular traffic, as a mutant Mon1a allele resulted in increased localization of cell surface proteins, whereas reduced levels of Mon1a showed decreased secretory activity. Here we show that Mon1a affects different steps in the secretory pathway including endoplasmic reticulum-to-Golgi traffic. siRNA-dependent reduction of Mon1a levels resulted in a delay in the reformation of the Golgi apparatus after Brefeldin A treatment. Endoglycosidase H treatment of ts045VSVG-GFP confirmed that knockdown of Mon1a delayed endoplasmic reticulum-to-Golgi trafficking. Reductions in Mon1a also resulted in delayed trafficking from Golgi to the plasma membrane. Immunoprecipitation and mass spectrometry analysis showed that Mon1a associates with dynein intermediate chain. Reductions in Mon1a or dynein altered steady state Golgi morphology. Reductions in Mon1a delayed formation of ERGIC-53-positive vesicles, whereas reductions in dynein did not affect vesicle formation. These data provide strong evidence for a role for Mon1a in anterograde trafficking through the secretory apparatus.

First Report of Pepper mottle virus Infecting Tomato in Hawaii

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5'- CACTCCCTATTATCCAGG(T)16-3') following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States.

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

Accuracy and Precision of the Pronto-7 for Noninvasive Hemoglobin Testing in An Outpatient Setting

Abstract 3147 IntroductionInvasive blood draw and laboratory analysis for determination of total hemoglobin concentration (tHb) is routine in the outpatient setting. However, the method is time consuming, uncomfortable for the patient, and exposes caregivers to risks associated with needle sticks and blood exposure. In contrast, hemoglobin determination with Pulse CO-Oximetry (SpHb) allows for noninvasive and quick estimation of hemoglobin with a spectrophotometric finger sensor. Continuous SpHb has been validated in the ICU [1], surgery[2], and volunteers undergoing hemodilution, [3] but the accuracy of “spot check” SpHb devices has not been validated in the outpatient setting. Our objective was to compare spot-check SpHb and invasive point-of-care (POC) hemoglobin testing to tHb measurements from a laboratory hematology analyzer. MethodsStudy subjects were adult and pediatric subjects presenting to two outpatient clinics and one health screening event. A noninvasive hemoglobin test device (Pronto-7, software version 2.19, Masimo Corp. Irvine CA) and reusable finger clip sensor (rainbow 4D) was placed on the patient's non-dominant ring finger. The sensor was covered with an opaque shield to prevent optical interference. Two consecutive SpHb readings were obtained from each subject, detaching and reattaching the sensor between each measurement. Testing was conducted while patients were quiet and sitting upright. A venous blood sample was obtained by venipuncture of the median cubital vein of the non-dominant arm and a capillary sample was obtained by finger stick according to the manufacturer's directions for use. The venous sample was analyzed for reference tHb with a laboratory hematology analyzer (LH 780, Beckman Coulter, USA). The capillary sample was analyzed immediately following the finger stick with a POC device (HemoCue 201+, Hemocue, Cypress CA). The SpHb and POC tHb were compared to the laboratory tHb. Bias, standard deviation, and coefficient of variation (precision) were calculated. ResultsA total of 474 subjects (37% male, 39 +/− 17 years of age) were enrolled, 431 (91%) from outpatient clinics and 43 (9%) from a community screening event. Subjects consisted of 246 (52%) healthy volunteers and 228 (48%) with one or more of the following conditions: high blood pressure, diabetes, high cholesterol, chronic obstructive pulmonary disease, or pregnancy. A total of 399 (84%) had light pigmentation, 71 (15%) medium pigmentation, and 3 (1%) had dark pigmentation. Reference tHb ranged from 8.6 to 17.4 g/dL. There were 877 SpHb measurements, 443 POC tHb measurements, and 473 reference tHb measurements. The mean bias and standard deviation of SpHb was −0.1 +/− 1.1 g/dL and of POC tHb was -0.1 and 1.6 g/dL. Using the first and second SpHb measurements, the precision of SpHb was 4.1%. ConclusionNoninvasive hemoglobin testing with the Pronto-7 had acceptable accuracy with similar bias and lower standard deviation than a widely available, POC tHB test device. The Pronto-7 has the potential to enhance patient care and caregiver safety in the outpatient setting. Disclosures:Shah:Masimo Corp.: Consultancy, Honoraria, Speakers Bureau. Martinez:Masimo Corp.: Research Funding. Osea:Masimo Corp.: Research Funding.

Effect of Hydroxyapatite and Bovine Serum Albumin on the Antibacterial Activity of MTA.

The purpose of this study was to compare the inhibitory effect of bovine serum albumin (BSA) and hydroxyapatite (HA) on the antibacterial activity of white-colored MTA (WMTA) against Staphylococcus (S.) aureus and Streptococcus (S.) mutans after 24 and 72 hours. All materials were prepared according to the manufacturer's directions immediately before testing. The antibacterial effect of each group (WMTA, WMTA+BSA and WMTA+HA) was determined by measuring the diameter of zone of inhibition in millimeters after incubation at 37°C for 24 and 72 hours in a humid atmosphere. Each test was repeated three times. Data were analyzed using ANOVA and Tukey's test. In the 24 hours samples as well as in 72 hours samples, the antibacterial activity of MTA+HA group was significantly greater than two other groups against S. aureus (P<0.05). However, the antibacterial activity of MTA+HA group against S. mutans was not significantly different from the MTA group in 24 hours as well as 72 hours samples. BSA reduced the antibacterial activity of MTA against both tested bacteria in the 24 and 72 hour samples (P<0.05). The products studied exhibited antibacterial activity. However, in both time intervals, the MTA+HA group exerted the greatest activity against S. mutans and S. aureus.

Impact of Cleaning Regimens in Silver-Impregnated and Hydrogen Peroxide Lens Cases

Lens storage case hygiene practices are important for safe contact lens wear. However, the effectiveness of the manufacturer's direction for use and various cleaning regimens in reducing biofilm load is yet to be evaluated and compared. This in vitro study compared the effectiveness of several cleaning methods using silver-impregnated lens case and hydrogen peroxide disinfection systems. Biofilms of Pseudomonas aeruginosa 122 and Staphylococcus aureus ATCC 6538 were grown in silver-impregnated and hydrogen peroxide lens cases. After the establishment of the biofilms, the silver-impregnated case was subjected to one of four cleaning regimens: "manufacturer's directions for use--rinsed and recapped"; "rubbed, rinsed, and recapped"; "rubbed, rinsed, and air dried"; or "rubbed, rinsed, tissue wiped, and air dried." Hydrogen peroxide cases underwent one of two regimens: "manufacturer's directions for use--rinsed with saline and air dried" or "soaked in solution for 6 hrs." The level of residual bacteria was quantified. The efficacy of each cleaning regimen was then compared. Mechanical rubbing and wiping of silver-impregnated cases and soaking hydrogen peroxide cases in hydrogen peroxide were the most effective treatments for reducing biofilms. Once the silver-impregnated cases were rubbed, air drying or recapping the cases did not have any significant effect on the level of the biofilm. The level of initial biofilm formation of silver-impregnated cases was significantly lower than those of polypropylene lens cases (P<0.0001). Rubbing and rinsing with disinfecting solution and wiping with a tissue can be considered to be effective in removing biofilms from silver-impregnated lens cases. Resoaking the basket-type lens case in hydrogen peroxide between use was found to be effective in removing biofilms from these cases.

MYC Protein Inhibits Transcription of the MicroRNA Cluster MC-let-7a-1∼let-7d via Noncanonical E-box

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

N-Acetylfarnesylcysteine Is a Novel Class of Peroxisome Proliferator-activated Receptor γ Ligand with Partial and Full Agonist Activity in Vitro and in Vivo

The thiazolidedione (TZD) class of drugs is clinically approved for the treatment of type 2 diabetes. The therapeutic actions of TZDs are mediated via activation of peroxisome proliferator-activated receptor γ (PPARγ). Despite their widespread use, concern exists regarding the safety of currently used TZDs. This has prompted the development of selective PPARγ modulators (SPPARMs), compounds that promote glucose homeostasis but with reduced side effects due to partial PPARγ agonism. However, this also results in partial agonism with respect to PPARγ target genes promoting glucose homeostasis. Using a gene expression-based screening approach we identified N-acetylfarnesylcysteine (AFC) as both a full and partial agonist depending on the PPARγ target gene (differential SPPARM). AFC activated PPARγ as effectively as rosiglitazone with regard to Adrp, Angptl4, and AdipoQ, but was a partial agonist of aP2, a PPARγ target gene associated with increased adiposity. Induction of adipogenesis by AFC was also attenuated compared with rosiglitazone. Reporter, ligand binding assays, and dynamic modeling demonstrate that AFC binds and activates PPARγ in a unique manner compared with other PPARγ ligands. Importantly, treatment of mice with AFC improved glucose tolerance similar to rosiglitazone, but AFC did not promote weight gain to the same extent. Finally, AFC had effects on adipose tissue remodeling similar to those of rosiglitazone and had enhanced antiinflammatory effects. In conclusion, we describe a new approach for the identification of differential SPPARMs and have identified AFC as a novel class of PPARγ ligand with both full and partial agonist activity in vitro and in vivo.

Nanoleakage at the resin-dental interface of four self-etching adhesives

To evaluate the nanoleakage and ultramorphology of four self-etching adhesives. Sixteen freshly extracted, caries-free human third molars were selected. A flat dentin surface was exposed by removing occlusal enamel. All teeth were randomly divided into four groups acorrding to four different self-etch adhesive: Adper Prompt (A), iBond (B), Xeno III (C) and SE Bond (D). The dentin were bonded with dentin adhesive system according to manufacturer's directions. Composite layers were built up incrementally. The specimens were sectioned longitudinally across the resin-dentin interface into 4.0 mm×0.9 mm sticks and then traced with ammoniacal silver solution. Epoxy resin-embedded sections were prepared for transmission electron microscope (TEM) to observe nanoleakage. The images were qualitatively compared by NIH software, and data was analyzed by SPSS. Different thickness of hybrid layer and adhesives layer were observed for each adhesive. The hybrid layer of A, C was thicker than that of B, D, and adhesive layer of D was thicker than the others. The extent of nanoleakage varied among different adhesives: A (45.02 ± 9.49), B (43.97 ± 8.55), C (27.02 ± 10.86), D (12.94 ± 2.07). D presented significantly less silver deposition than any of the others did (P < 0.05). The thickness of hybrid layer and adhesive layer vary among the four adhesives. The shape and extent of nanoleakage of each adhesive are also different. Two-step system shows less nanoleakage than one-step systems do.

Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

HSP70 Protein Promotes Survival of C6 and U87 Glioma Cells by Inhibition of ATF5 Degradation

Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.

Effect of Light Activation on Tooth Sensitivity After In-Office Bleaching

This clinical study evaluated the effects of light-emitting diode (LED)/laser activation on bleaching effectiveness (BE) and tooth sensitivity (TS) during in-office bleaching. Thirty caries-free patients were divided into two groups: light-activated (LA) and non-activated (NA) groups. A 35% hydrogen peroxide gel (Whiteness HP Maxx, FGM Dental Products, Joinville SC, Brazil) was used in three 15-minute applications for both groups. For the LA group, LED/laser energy (Whitening Lase Light Plus, DMC Odontológica, São Carlos SP, Brazil) was used, in accordance with the manufacturer's directions. Two sessions of bleaching were performed at one-week intervals. Color was registered at baseline and after the first and second bleaching sessions using a Vita shade guide. Patients recorded TS on a 0 to 4 scale during bleaching and within the next 24 and 48 hours of each session. BE at recall each week and intensity of TS were evaluated by repeated measures analysis of variance (ANOVA) and Tukey tests (α=0.05). Tooth sensitivity was compared using the Friedman repeated measures analysis of variance by rank and the Wilcoxon sign-ranked test. Faster bleaching was observed for the LA group than for the NA group after the first session (4.8 and 3.8 shade guide units [SGUs]; p=0.0001). However, both techniques were capable of bleaching the same number of SGUs after the second bleaching session (p=0.52). Most of the LA group (53.3%) had sensitivity even 24 hours after each bleaching session, but only 26.6% from the NA group reported TS. The intensity of TS was similar for both groups immediately after bleaching but significantly higher for the LA group 24 hours after each bleaching session (p=0.001). After two bleaching sessions, the use of LED/laser light activation did not improve bleaching speed. Persistent tooth sensitivity and higher tooth sensitivity after 24 hours of bleaching were observed when light activation was used.

Combination Treatment with HER-2 and VEGF Peptide Mimics Induces Potent Anti-tumor and Anti-angiogenic Responses in Vitro and in Vivo

HER-2 is a member of the EGF receptor family and is overexpressed in 20-30% of breast cancers. HER-2 overexpression causes increased expression of VEGF at both the RNA and protein levels. HER-2 and VEGF are therefore considered good targets for cancer treatment, which has led to the development of two humanized monoclonal antibodies (mAb) pertuzumab and bevacizumab. Although passive immunotherapy with these Abs are approved for treatment of advanced breast cancer, a number of concerns exist. Treatment is expensive, has a limited duration of action, and is usually accompanied by serious side effects. We hypothesized that therapy with conformational peptide mimics aimed at blocking receptor-ligand interaction is potentially safer with little toxicity, cheaper with a longer half-life, and has greater penetrating abilities than mAbs. We designed and synthesized peptides based on the binding of HER-2 with pertuzumab and VEGF with VEGFR2. We show that treatment with the peptide mimics induces potent anti-tumor responses in vitro as determined by cell viability, proliferation, and HER2 phosphorylation assays. We also demonstrate in a transplantable BALB/c mouse tumor model that treatment with the peptide mimics resulted in a greater delay in tumor growth and development. Similarly, treatment with the peptide mimics inhibited angiogenesis in vivo as assessed by a Matrigel plug assay. To address the problem of degradability of L-amino acid peptides in vivo, we synthesized the retro-inverso D-peptide mimics that resulted in higher efficacy in treatment. Our study shows that combination treatment with HER-2 and VEGF peptide mimics provides greater efficacy than individual treatments.