David J Kolodrubetz

Evaluation of Ultrasonically Placed MTA and Fracture Resistance with Intracanal Composite Resin in a Model of Apexification

The purpose of this study was to evaluate whether intracoronal delivery of an apical barrier of mineral trioxide aggregate (MTA) placed ultrasonically, non-ultrasonically, or ultrasonically with the addition of an intracanal composite resin provided a better seal against bacterial leakage. A second purpose was to determine whether intracanal composite resin or gutta-percha and sealer placed against an apical barrier of MTA provided greater resistance to root fracture. In a standardized in vitro open apex model, MTA was placed as an apical barrier at a thickness of 4 mm, with and without ultrasonic vibration. The barriers were challenged with bacteria exposure within a leakage model, and fracture resistance was assessed with increasing forces applied via an Instron machine. After 45 days, the addition of ultrasonics significantly improved the MTA seal, compared with the non-ultrasonics treatment (Kruskal Wallis nonparametric ANOVA with Dunn multiple comparison test p < 0.05). Bacterial leakage occurred in 6 (33%) of 18 in the non-ultrasonic MTA group, 2 (11%) of 18 in the ultrasonic MTA group, and 1 (6%) of 18 in the ultrasonic MTA-composite group. There were no significant differences at 90 days. A 4-mm thickness of MTA followed with an intracanal composite resin demonstrated a significantly greater resistance to root fracture than MTA followed with gutta-percha and sealer (one-way ANOVA with Newman-Keuls multiple comparison test, p < 0.01). The MTA-gutta-percha group was not significantly different than the MTA unrestored positive control.

NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.

The yeast SLK1 (BCK1) gene encodes a mitogen-activated protein kinase (MAPK) activator protein which functions upstream in a protein kinase cascade that converges on the MAPK Slt2p (Mpk1p). Dominant alleles of SLK1 have been shown to bypass the conditional lethality of a protein kinase C mutation, pkc1-delta, suggesting that Pkc1p may regulate Slk1p function. Slk1p has an important role in morphogenesis and growth control, and deletions of the SLK1 gene are lethal in a spa2-delta mutant background. To search for genes that interact with the SLK1-SLT2 pathway, a synthetic lethal suppression screen was carried out. Genes which in multiple copies suppress the synthetic lethality of slk1-1 spa2-delta were identified, and one, the NHP6A gene, has been extensively characterized. The NHP6A gene and the closely related NHP6B gene were shown previously to encode HMG1-like chromatin-associated proteins. We demonstrate here that these genes are functionally redundant and that multiple copies of either NHP6A or NHP6B suppress slk1-delta and slt2-delta. Strains from which both NHP6 genes were deleted (nhp6-delta mutants) share many phenotypes with pkc1-delta, slk1-delta, and slt2-delta mutants. nhp6-delta cells display a temperature-sensitive growth defect that is rescued by the addition of 1 M sorbitol to the medium, and they are sensitive to starvation. nhp6-delta strains also exhibit a variety of morphological and cytoskeletal defects. At the restrictive temperature for growth, nhp6-delta mutant cells contain elongated buds and enlarged necks. Many cells have patches of chitin staining on their cell surfaces, and chitin deposition is enhanced at the necks of budded cells. nhp6-delta cells display a defect in actin polarity and often accumulate large actin chunks. Genetic and phenotypic analysis indicates that NHP6A and NHP6B function downstream of SLT2. Our results indicate that the Slt2p MAPK pathway in Saccharomyces cerevisiae may mediate its function in cell growth and morphogenesis, at least in part, through high-mobility group proteins.

Nhp6, an HMG1 Protein, Functions in SNR6 Transcription by RNA Polymerase III in S. cerevisiae

Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.

Regulation of the l-arabinose transport operons in Escherichia coli☆

l-Arabinose is transported into Escherichia coli via two independent transport systems, a system possessing relatively low affinity for arabinose, the araE system, and a system of higher affinity for arabinose, the araFG system. In the work reported here we demonstrate that insertion of the Mu-lac bacteriophage isolated by Casadaban & Cohen (1979) permits a reliable measurement of the expression of these two operons. Using appropriate Mu-lac insertion strains we found that both of the arabinose transport operons can be induced approximately 150-fold by the presence of arabinose, and that induction of both transport operons requires CRP (cyclic AMP receptor protein), but that their catabolite sensitivities differ from one another. In addition, we show that the araC+ allele is dominant to the Cc allele in the control of the transport operons, just as is found in the araBAD operon.

The Regulation of Leukotoxin Production in Actinobacillus Actinomycetemcomitans Strain JP2

Actinobacillus actinomycetemcomitans (A.a.) can produce a potent leukotoxin that is thought to be involved in evasion of the host immune response. In order to understand the role of A.a. and its leukotoxin in the initiation and progression of periodontal disease, it is important determine how the expression of A.a. virulence factors might be regulated by the local periodontal micro-environment. To facilitate the measurement of leukotoxin levels, a leukotoxin-β-galactosidase gene fusion was constructed and recombined into the chromosome of A.a. strain JP2 at the leukotoxin locus. The resulting strain, AAM17, produces β-galactosidase under control of the leukotoxin promoter. It also produces leukotoxin, since integration of the gene fusion into the chromosome was designed to produce a duplication of the leukotoxin gene. This strain was used to measure the change in leukotoxin level in response to alterations in two environmental signals: iron concentration and oxygen tension. When AAM17 was grown in iron-limited media that did not alter growth rate but did increase the levels of other iron-regulated proteins, the levels of the leukotoxin-p-galactosidase were similar to those found in AAM17 grown in iron-replete media. These results were confirmed in strains AAM17 and JP2 by leukotoxicity assays and RNA blots. Aerobic growth of AAM17 resulted in a three-fold decrease in leukotoxin β-galactosidase activity compared with anaerobically grown cells. These results indicate that the A.a. leukotoxin is regulated by some of the environmental signals that may vary in the gingival crevice.

Use of Defined Mutants To Assess the Role of the Campylobacter rectus S-Layer in Bacterium-Epithelial Cell Interactions

ABSTRACT Campylobacter rectus is a periodontal pathogen with a 150-kDa protein on its cell surface. This protein forms a paracrystalline lattice, called the S-layer, surrounding the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the possible role of the S-layer in the initial interaction of C. rectus with host epithelial cells, C. rectus strains lacking the S-layer protein gene (crsA) were constructed by allelic exchange mutagenesis. Surprisingly, the lack of the S-layer had only a minor effect on the interaction of C. rectus with HEp-2 epithelial cells; CrsA+ cells were 30 to 50% more adherent than were CrsA− bacteria. Since the host cell expression of cytokines appears to play an important role in the pathogenesis of periodontal diseases, the effect of the S-layer on the epithelial cell cytokine response was also examined by quantitative reverse transcriptase PCR and enzyme-linked immunosorbent assay. Although there were no changes in the mRNA levels for the anti-inflammatory cytokines interleukin-1 receptor agonist (IL-1ra), IL-13, and transforming growth factor β, the expression and secretion of the proinflammatory cytokines IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) were significantly induced by both wild-type C. rectus and CrsA− bacteria. Interestingly, the kinetics of cytokine induction differed for the CrsA+ and CrsA−bacteria. At early time points, the HEp-2 cells challenged with CrsA− bacteria produced higher levels of IL-6, IL-8, and TNF-α mRNA and protein than did cells challenged with CrsA+ bacteria. We conclude that C. rectus may help initiate periodontitis by increasing the expression of proinflammatory cytokines and that the S-layer may temper this response to facilitate the survival of C. rectus at the site of infection.

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, γ-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (kcat/Km of 8.2 μm–1 min–1) not l-Leu-p-NA (kcat/Km of 1.1 μm–1 min–1). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, γ-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.

Cloning and Expression of Two Novel Hemin Binding Protein Genes from Treponema denticola

ABSTRACT Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed,T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of thehbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in severalT. denticola ATCC strains and clinical isolates, but not inTreponema pectinovorum, Treponema socranskii, orEscherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, calledhbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. ThehbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein.

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.

Amino-terminal sequence of a Saccharomyces cerevisiae nuclear protein, NHP6, shows significant identity to bovine HMG1

Several nonhistone chromatin proteins (NHPs) have been isolated from Saccharomyces cerevisiae nuclei. They have molecular masses and amino acid compositions typical of the high mobility group (HMG) proteins from higher eukaryotic cells. Polyclonal antisera raised against two of the NHPs have been used in immunoblots of proteins from subcellular fractions of yeast to show that the NHPs are indeed nuclear. In addition, the amino‐terminal amino acid sequences of several of the NHPs were determined. Importantly, the amino‐terminal sequence of one of the proteins, NHP6, has significant (60%) identity with a stretch of amino acids in calf thymus HMG1.