In vitro staining effects of stannous fluoride and sodium fluoride on ceramic material.

STATEMENT OF PROBLEM: Long-term fluoride application on the teeth of patients receiving radiation therapy for head and neck tumors results in excessive staining and roughening of ceramic restorations. PURPOSE: The purpose of this in vitro study was to compare the staining effects of 2 fluoride treatments on ceramic disks by simulating 1 year of clinical exposure at 10 minutes per day. In addition, 2 different surface preparations were tested. MATERIAL AND METHODS: Eighty ceramic disks (IPS Empress), 20 x 2 mm, were fabricated. Half of the disks were glazed, and the remaining disks were polished. All disks were brushed for 3 minutes with a soft-bristle power toothbrush and mild dentifrice (baseline) and were immersed in 1 of the 2 fluoride products (0.4% SnF(2), Gel-Kam Gel, or 1.1% NaF, Prevident 5000) for 10 days (n=20). Means and standard deviations of color change (Delta E), surface roughness (Ra, um), and surface gloss (GU) of the ceramic material were measured with a reflection spectrophotometer, a profilometer, and a gloss meter, respectively, at baseline and after fluoride treatment. Two- and 3-way ANOVA (alpha=.05), with surface preparation (polished vs. glazed) and fluoride treatment (0.4% SnF(2) or 1.1% NaF) as independent variables and condition (baseline vs. after fluoride treatment) as a repeated measure, was used to analyze the data. Fisher's PLSD intervals (alpha=.05) were calculated for comparisons among the means. RESULTS: The polished specimens had significantly higher Delta E values, significantly higher surface gloss values, and significantly lower surface roughness values than the glazed specimens before fluoride treatment (P<.001). After both fluoride treatments, ceramic disks exhibited significantly higher surface roughness values when polished and significantly lower surface gloss values when glazed or polished (P<.001). The glazed specimens presented significantly higher surface roughness (P<.001) and lower surface gloss values (P<.001) when treated with 0.4% SnF(2) as compared to NaF. For the polished specimens, there was no significant difference in surface roughness and surface gloss values between the 2 fluoride treatments. CONCLUSIONS: Use of 0.4% SnF(2) and 1.1% NaF gels, in vitro, caused significant color change in the polished IPS Empress ceramic disks. Polishing of the ceramic surface before immersion in either fluoride agent caused the ceramic tested to be more resistant to etching by the 2 solutions tested. The NaF caused less deterioration of the porcelain surface and was less stain inducing than SnF(2).

In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli.

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Genetic and physical analysis of the bacteriophage P22 tail protein

A plasmid based genetic system was developed for the tail protein of the Salmonella typhimurium bacteriophage P22 and used to isolate and characterize tail protein mutants. The tail protein is a trimeric structural protein of the phage and an endorhamnosidase whose activity is essential for infection. The gene for the tail protein has previously been cloned into a plasmid expression vector and sequenced. A plate complementation assay for tail protein produced from the cloned gene was developed and used to isolate 27 tail protein mutants following mutagenesis of the cloned gene. These mutations were mapped into 12 deletion intervals using deletions which were made on plasmids in vitro and crossed onto P22. The base substitutions were determined by DNA sequencing. The majority of mutants had missense or nonsense mutations in the protein coding portion of the gene; however four of the mutants were in the putative transcription terminator. The oligomeric state of tail protein from the 15 missense mutants was investigated using SDS and nondenaturing polyacrylamide gel electrophoresis of cell lysates. Wild-type tail protein retains its trimeric structure in SDS gels at room temperature. Two of the mutant proteins also migrated as trimers in SDS gels, yet one of these had a considerably faster mobility than wild-type trimer. Its migration was the same as wild-type in a nondenaturing gel, so it is thought to be a trimer which is partially denatured by SDS. Four of the mutants produced proteins which migrate at the position of a monomer in an SDS gel but cannot be seen on a nondenaturing gel. These proteins are thought to be either monomers or soluble aggregates which cannot enter the nondenaturing gel. The remainder of mutants produce protein which is degraded. The mutant tail protein which had normal trimeric mobility on SDS and nondenaturing gels was purified. This protein has essentially wild-type ability to attach to phage capsids, but its endorhamnosidase activity is only 4% of wild-type. ^

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

CHARACTERIZATION OF HIGH MOBILITY GROUP AND HISTONE H1 PROTEIN LEVELS AND SYNTHESIS DURING SPERMATOGENESIS IN THE RAT

In order to propose a role for internucleosomal high mobility group proteins (HMGs), and HI histone variants study of their levels and synthesis in a system of development and differentiation--rat spermatogenesis--was undertaken. HMG1, 2, 14, and 17 were isolated from rat testes and found to be very similar to calf thymus HMGs. Testis levels of HMGs, relative to DNA, were equivalent to other rat tissues for HMG1 (13 ug/mg DNA), HMG14 (2 ug/mg DNA), and HMG17 (5 ug/mg DNA). HMG2 levels were different among rat tissues, with three groups observed: (1) nonproliferating tissues (1-5 ug/mg DNA); (2) proliferating tissues (8-13 ug/mg DNA); and (3) the testis (32 ug/mg DNA). Other species (toad, opposum, mouse, dog, and monkey) showed the same testis-specific increase of HMG2. Populations of purified testis cell types were separated by centrifugal elutriation and density gradient centrifugation from adult and immature rat testes. Pachytene spermatocytes and early spermatids (56 and 47 ug/mg DNA, respectively) caused the testis-specific increase of HMG2 levels. Cell types preceding pachytenes (types A and B spermatogonia, mixtures of spermatogonia and early primary spermatocytes, and early pachytenes contained HMG2 levels similar to proliferating tissues (12 ug/mg DNA). Late spermatids did not contain HMGs. Somatic Sertoli and Leydig cells (2 ug/mg DNA) exhibited HMG2 levels similar to nonproliferating tissues. HMGs synthesized in spermatogonia and spermatocytes had similar specific activities, but early spermatids did not synthesize HMGs. Germ cells also contained an HMG2 species (on acid-urea gels) not found in somatic tissues. Other investigators have shown that HMGs may be associated with transcriptional or replicative processes. Thus, it is proposed that HMG2 plays a role in modulatable gene expression, while HMG1 is associated with housekeeping functions.^ HI histone variants were also studied throughout spermatogenesis. The minor somatic variant, HIa, is the predominant variant in spermatogonia and early primary spermatocytes. In early pachytenes, the testis-specific variant, HIt, is first synthesized and appears, largely replacing somatic variants HIbcd and e by late pachytene stage. Early spermatids contain the same HI composition as pachytenes, but do not synthesize HI histones. HI('0) is present in low amounts in all germ cells. These results suggest that expression of HI variants is developmentally controlled.^

ISOELECTRIC FOCUSING STUDIES OF STEROID BINDING PROTEINS

Steroid binding proteins are an obvious choice in the search for genetic factors in plasma that might predispose to upper body obesity, a risk factor for non-insulin dependent diabetes and cardiovascular disease. The two steroid binding proteins studied by isoelectric focusing were sex hormone binding globulin (SHBG), the transport protein for sex hormones and corticosteroid binding globulin (CBG), the transport protein for corticosteroids. Auto-radiography and immunoblotting on polyacrylamide gels were used to detect polymorphism in SHBG. Immunoblotting on agarose gels was used to visualize corticosteroid binding globulin. SHBG showed similar structural variation in American Caucasians, American Blacks and Canadian Indians. Two alleles (1, 2) were hypothesized with highly polymorphic frequencies in all three ethnic groups. CBG was not found to be polymorphic, but two variants were found in Caucasian male twins and in a Black individual. The finding of a good assay and a polymorphic system for SHBG are the first steps for additional studies into disease associations. ^

Regulation of tyrosine hydroxylase gene expression by mRNA stabilization

Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 μM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3′UTR using RNA electro-mobility shift assays show the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains two proteins. Northwesterns yielded similar results. Transfection studies reveal an increase in expression of the full-length TH transcript due to DMPP-treatment similar to that of endogenous TH mRNA. This finding suggests the increased expression is due primarily to mRNA stabilization. Transfection of luciferase reporter constructs containing regions of the TH 3′UTR reveal only the full-length 3′UTR influenced the expression level of reporter transcripts. ^