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PubMed1990

An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.

Amino Acid Sequence↗
PubMed1994

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.

Animals↗
PubMed2006

New concept of resin-dentin interfacial adhesion: the nanointeraction zone.

The purpose of this study was to evaluate the morphological appearance of resin-dentin interface produced by 1-step adhesive systems in order to classify them, using TEM. It was hypothesized that all interfaces produced by each 1-step adhesive show characteristics similar to that produced by the conventional self-etching system rather than that produced by etch-and-rinse system. The flattened dentin surfaces were subjected to bonding treatment using either a 2-step adhesive: single bond, mega bond, unifil bond; or a 1-step adhesive: Prompt L-Pop, absolute, xeno CF, AQ bond, one-up bond F, G bond, and SSB-200. A flowable resin was then applied to these surfaces. The results revealed that resin-dentin interfaces produced by 1-step systems were classified into three categories. Additionally, the interface produced by G bond and SSB-200 showed a different morphological appearance from that of the others. The interface produced by these adhesives was thinner and less demineralized, with the presence of hydroxyapatite-coated collagen fibrils. It is proposed that "Nanointeraction Zone (NIZ)" is a new complementary classification for partially demineralized resin-dentin interdiffusion zones. Thus, one-step self-etching adhesive systems can be classified into three categories, etch-and-rinse type, mild self-etching type, and NIZ type, by their morphological appearance of the interface.

Bisphenol A-Glycidyl Methacrylate↗
PubMed2006

Solid-state (14)N MAS NMR of ammonium ions as a spy to structural insights for ammonium salts.

The high resolution offered by magic-angle spinning (MAS), when compared to the static condition in solid-state NMR of powders, has been used to full advantage in a (14)N MAS NMR study of some ammonium salts: CH(3)NH(3)Cl, (NH(4))(2)(COO)(2) x H(2)O, (CH(3))(3)(C(6)H(5)CH(2))NCl, (CH(3))(3)(C(6)H(5))NI, [(n-C(4)H(9))(4)N](2)Mo(2)O(7), (NH(4))(2)HPO(4), and NH(4)H(2)PO(4). It is shown that the high-quality (14)N MAS NMR spectra, which can be obtained for these salts, allow determination of the (14)N quadrupole coupling parameters, i.e. C(Q) (the quadrupole coupling constant) and eta(Q) (the asymmetry parameter), with very high precision. In particular, it is shown that precise C(Q), eta(Q) parameters can be determined for at least two different (14)N sites in case the individual spinning-sideband (ssb) intensities arise from a single manifold of ssbs, i.e. the ssbs for the two sites cannot be resolved. This feature of (14)N MAS NMR, which is the first demonstration for manifolds of ssb in MAS NMR without the potential information from a central transition, becomes especially useful at the slow spinning frequencies (nu(r) = 1000-1500 Hz) applied to some of the ammonium salts studied here. The detection of the number of sites has been confirmed by the corresponding crystal structures determined from single-crystal X-ray diffraction (XRD), either in this work for the unknown structure of benzyl trimethylammonium chloride or from reports in the literature. The magnitudes of the (14)N quadrupole coupling constants for the ammonium salts studied here are in the range from C(Q) approximately 20 kHz to 1 MHz while the asymmetry parameters span the full range 0 < or = eta(Q) < or = 1. Clearly, the (14)N quadrupole coupling parameters (C(Q), eta(Q)) for ammonium ions appear highly sensitive toward crystal structure and therefore appreciably more informative for the characterization of ammonium salts in comparison to the isotropic (14)N (or (15)N) chemical shifts.

Journal Article↗
PubMed2003

Positive salivary gland biopsy, Sjögren syndrome, and neuropathy: clinical implications.

The relationship between neuropathy and Sjögren syndrome has been predicated largely on sicca symptoms or serological abnormalities rather than salivary gland pathology. We reviewed consecutive neuropathy patients who had had a lip biopsy to identify features of the neuropathy that were associated with a positive lip biopsy suggesting Sjögren syndrome. Twenty of 54 neuropathy patients were biopsy positive; 13 had a painful or nonspecific sensory neuropathy and only 4 were ataxic. Sicca symptoms were not associated with a positive biopsy (P = 0.14). Serological abnormalities were found more often in the biopsy-positive group (P = 0.008), but anti-Sjögren syndrome A or B (anti-SSA or SSB) antibodies were detected in only 30%. There were no other clinical or electromyographic (EMG) features associated with a positive biopsy. From this experience, we conclude that: (1). most patients with neuropathy and a positive lip biopsy for Sjögren syndrome have a painful, distal, sensory axonal neuropathy; (2). there are no clinical or EMG features that are predictive of a positive lip biopsy; (3). ataxic neuropathy is uncommon; and (4). the lack of sicca symptoms or anti-SSA or SSB antibodies in patients with neuropathy does not exclude Sjögren syndrome based upon salivary gland pathology.

Adult↗
PubMed1994

Successful outcome of pregnancy after treatment of maternal anti-Ro (SSA) antibodies with immunosuppressive therapy and plasmapheresis.

In this short communication we describe a patient with anti-SSA and anti-SSB antibodies whose first child died of congenital complete heart block (CCHB). During her second pregnancy she was treated with prednisolone, azathioprine, and plasmapheresis, and levels of anti-SSA and anti-SSB antibodies fell significantly. The pregnancy evolved uneventfully and resulted in the birth of an unaffected male infant. This is the fourth reported case of a successful outcome of pregnancy after treatment with immunosuppressive drugs in a woman with a significant risk of recurrence of CCHB.

Adult↗
PubMed2003

Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA.

In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.

Adenosine Triphosphate↗
PubMed1995

Growth inhibition and cell killing by N-methyl-N-nitrosourea: metabolic alterations that accompany poly(ADP-ribosyl)ation.

The effect of inhibition of poly(ADP-ribose) polymerase (PARP) on the growth arrest and cell killing induced by N-methyl-N-nitrosourea (MNU) was studied in L929 fibroblasts. Depletion of NAD and ATP preceded the cell killing by a 1-h exposure to 10 or 15 mM MNU. 3-Aminobenzamide (ABA), an inhibitor of PARP, spared the depletion of NAD and ATP and prevented the cell killing. With 5 mM MNU, a depletion of NAD was promptly reversed, and there was no loss of ATP and no cell death. Aphidicolin, a DNA polymerase inhibitor, prevented the restoration of NAD, with resulting depletion of ATP and death of the cells, effects that were prevented by ABA. Azide together with 2-deoxyglucose depleted ATP, followed by a loss of NAD and cell death, changes that occurred in the absence of DNA single strand breaks (DNA SSB). ABA prevented the depletion of NAD, but not that of ATP, nor the cell killing. MNU (2.5 mM) inhibited cell growth without effect on the viability of the cells. ABA potentiated the cell growth inhibition. Thus, inhibition of PARP potentiates cell growth inhibition by limiting DNA repair mechanisms. Alternatively, inhibition of the DNA repair response to more extensive DNA damage prevents cell killing. The ATP depletion caused by poly(ADP-ribosyl)ation, rather than DNA SSB and the loss of NAD, is the more critical event in the cell killing.

Adenosine Triphosphate↗
PubMed1996

Trolox inhibits peroxynitrite-mediated oxidative stress and apoptosis in rat thymocytes.

Peroxynitrite is a strong oxidant that reacts with a variety of biomolecules in vivo and in vitro. When rat thymocytes in phosphate buffer are exposed to 25 microM peroxynitrite for 10 min, DNA single strand breaks (SSB) can be detected. These SSB are repaired if the cells are incubated in fresh media at 37 degrees C for 120 min. In addition, DNA protein cross-links and apoptosis are observed 1 and 6 h, respectively, after peroxynitrite exposure. Peroxynitrite mediates the formation of thiobarbituric acid-reactive substances (TBARS) that may be responsible for the DNA-protein crosslinks (DPXL). Both TBARS and DPXL formation are lowered by posttreating the cells immediately after the 10-min exposure to peroxynitrite with Trolox, a water-soluble vitamin E analog. These results suggest that oxidative stress mediated by peroxynitrite can trigger a critical sequence of events ending in programmed cell death and that intracellular oxidation is a component of the apoptosis of thymocytes, since both oxidative processes and apoptosis can be prevented by Trolox. In addition to Trolox, we obtained partial data on three other phenolic antioxidants (3-tert-butyl-4-hydroxyanisole, butylated hydroxytoluene, and 2,6-diisopropylphenol). We find that Trolox and these three phenols similarly protect rat thymocytes from apoptosis mediated by peroxynitrite.

Animals↗
PubMed2000

Improved performance of pyrosequencing using single-stranded DNA-binding protein.

In modern biology, there is a critical need to develop a high-throughput and inexpensive platform for DNA sequencing. Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. In these studies, single-stranded DNA-binding protein (SSB) was added to the primed DNA template prior to the Pyrosequencing reaction. The addition of SSB to a Pyrosequencing reaction system resulted in a read length of more than 30 nucleotides. Improvements were observed as: (i) increased efficiency of the enzymes, (ii) reduced mispriming, as measured by nonspecific signals, (iii) an increase in signal intensity during the reaction, (iv) higher accuracy in reading the number of identical adjacent nucleotides in difficult templates, and (v) longer reads. The usefulness of these results for future Pyrosequencing applications is discussed.

Apyrase↗
PubMed1994

Synergistic cytotoxicity of carboplatin and divicine on murine erythroleukemic cells.

Carboplatin (CBDCA) and the pyrimidine aglycone divicine displayed cytotoxic effects of murine erythroleukemic cells (MELC), with ID50 values of 158 and 37 microM, respectively. Combination of CBDCA and divicine, at a 2:1 ratio, increased cytotoxicity considerably. Under specific conditions of time schedule of administration, the association of CBDCA and divicine resulted in a clear synergistic activity. Alkaline elution studies on both unirradiated and gamma-irradiated MELC demonstrated opposite patterns of DNA damage with the two molecules. Thus, CBDCA elicited DNA interstrand crosslinks (ISC), while divicine resulted in DNA single strand breaks (SSB). Association of both molecules led in the unirradiated cells to a higher SSB frequency than recorded with divicine alone. Accordingly, intracellular activation of CDBCA by redox cycling of divicine seems not to be involved. Rather, intracellular platinum appears to enhance cytotoxicity of divicine.

Animals↗
PubMed1996

Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs.

Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium. As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B. Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR. Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside. The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody. Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins. In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion. Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb. The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants. The recombinant B. abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs. Moreover, the results of this study suggest that B. abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.

Animals↗
PubMed2001

A novel dipolar dephasing method for the slow magic angle turning experiment.

Complete suppression of the resonances from protonated carbons in a slow magic angle spinning experiment can be achieved using five dipolar dephasing (Five-DD) periods distributed in one rotor period. This produces a spectrum containing only the spinning sidebands (SSB) from the nonprotonated carbons. It is shown that the SSB patterns corresponding to the nonprotonated carbons are not distorted over a wide range of dipolar dephasing times. Hence, this method can be used to obtain reliable principal values of the chemical shift tensors for each nonprotonated carbon. The Five-DD method can be readily incorporated into isotropic-anisotropic 2D experiments such as FIREMAT and 2D-PASS to facilitate the measurement of the (13)C chemical shift tensors in complex systems.

Carbon Isotopes↗
PubMed2001

Bond-shift rearrangement in solid Li(3)P(7)(monoglyme)(3): a (31)P MAS NMR study.

The (31)P MAS NMR spectrum of solid Li(3)P(7)(monoglyme)(3) has been reinvestigated over a wide temperature range (-70 to +77 degrees C) and under conditions of better resolution (Larmor frequency of 162 MHz and spinning rate of approximately 30 kHz) than previously measured (121 MHz and 13 kHz). At low temperatures three spinning sideband (ssb) manifolds are observed: a singlet (centered at -45 ppm relative to 85% H(3)PO(4)) due to the apical atom (A) of the P(7)-cage trianion; a 1 : 1 : 1 triplet (at -110, -117, and -124.5 ppm) due to the negatively charged equatorial (E) atoms, and a one to two doublet (at -161 and -168.5 ppm) due to the basal (B) atoms. These results are consistent with the P(7) cage having nearly, but not perfect, C(3v) symmetry. The compound appears to be well ordered in the solid state with very little structural dispersity. On heating, the NMR lines broaden and eventually coalesce into a single ssb manifold. This behavior is ascribed to bond-shift rearrangement similar to the Cope rearrangement in bullvalene. A MAS 2D exchange experiment and a quantitative analysis of the 1D NMR lineshapes indicate that, unlike in solution where the rearrangement involves a single bond shift at a time, in the solid the process involves a succession of two bond shifts: The first leads to an intermediate species in which the rearranged P(7) cage is inverted, while in the subsequent step a second bond shift takes place that also restores the original orientation of the cage in the lattice. The overall effect of the double bond shift is equivalent to cyclic permutation of the phosphorus atoms within the five member rings of the P(7)-cage. The quantitative analysis of the dynamic lineshapes shows that this cyclic permutation proceeds at a different rate in one ring (k(d)(1)) than in the other two (k(d)(2,3)). The kinetic parameters for these processes are E(a)(1)=18.7 kJ/mol, E(a)(2,3)=58.0 kJ/mol, k(d)(1)(17 degrees C)=k(d)(2,3)(17 degrees C)=10(4) s(-1). No indications for independent threefold molecular jumps of the P(7) cage were found.

Journal Article↗
PubMedDate not supplied

Elemental quantitation of natural organic matter by CPMAS 13C NMR spectroscopy.

Cross-polarized magic-angle-spinning NMR (CPMAS-NMR) techniques are assumed to be only semi-quantitative in the assessment of carbon distribution in humic substances or natural organic matter, due to a number of interferences such as spinning side bands (SSB) in spectra, paramagnetic species in samples, and low or remote protonation of aromatic carbons. Fast rotor spin rates or direct polarization NMR techniques are normally applied to improve quantitative signal detectability. Variable contact time pulse sequences were used here to obtain CPMAS-NMR spectra of organic compounds of known structure and different humic substances. Integration of spectral areas, previously subtracted of SSB, and relative stoichiometric factors were used for mathematical elaboration to calculate the elemental content in samples. These values did not significantly differ from those obtained by direct determination of elemental content with quantitative elemental analysis. Our results showed that the carbon observed CPMAS-NMR provides a quantitative representation of the whole carbon content in humic substances.

Magnetic Resonance Spectroscopy↗
PubMed1998

Induction of oxidative stress in brain tissues of mice after subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The ability of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce oxidative stress in hepatic and some extrahepatic tissues of animals is well documented. However, no previous study has examined the ability of TCDD to induce oxidative stress and tissue damage in brain in vivo. In this study the ability of TCDD to induce oxidative stress in brain tissues of mice was studied after subchronic exposures. Groups of female B6C3F1 mice were treated orally with TCDD (0, 0.45, 1.5, 15, and 150 ng/kg/day) for 13 weeks, 5 days/week. The animals were euthanized 3 days after the last treatment and brain tissues were collected. Biomarkers of oxidative stress including production of superoxide anion, lipid peroxidation, and DNA-single-strand breaks (SSB) were determined. TCDD treatment resulted in significant and dose-dependent increases in the production of superoxide anion as assessed by reduction of cytochrome c. Significant increases were also observed in lipid peroxidation and DNA-SSB in those tissues, as assessed by the presence of thiobarbituric acid-reactive substances and the alkaline elution technique, respectively. These results clearly indicate that subchronic exposure to low doses of TCDD can induce oxidative tissue damage in brain tissues which may at least in part play a role in the effects of TCDD on the central nervous system.

Animals↗
PubMed1986

Ultrafast chemical repair of DNA single and double strand break precursors in irradiated V79 cells.

The fast kinetics of reactions of free radical precursors of DNA single strand breaks (ssb) and double strand breaks (dsb) have been determined in Chinese hamster V79 cells by fast mixing and irradiation methods using the alkaline unwinding technique to assay breaks. Fast chemical repair of oxygen-dependent ssb and dsb precursors was observed and approached completion within 10-20 ms of irradiation. Treatment of cells with the glutathione synthesis blocking agent, buthionine sulphoximine, showed that approximately half of the chemical repair was attributable to intracellular non-protein thiols. The nature of the residual repair is obscure, but it is apparently not attributable to non-protein thiols. Similar repair rates and thiol dependences were also found for cell kill. With all three endopoints, oxygen competes with and blocks the chemical repair.

Animals↗
PubMed1996

Cytotoxic impact of DNA single vs double strand breaks in oxidatively injured cells.

Hydrogen peroxide is a potent inducer of DNA single strand breaks (SSBs) in cultured mammalian cells. These lesions, however, are efficiently repaired and do not appear to mediate the cytotoxic response. This inference is based on the observations that a) inhibiting the rate of SSB-removal does not result in an increased cytotoxicity; b) using different experimental conditions it is possible to dissociate the formation of DNA SSBs from the cytotoxic response; c) the induction/loss of the oxidant-resistant phenotype in cell variants characterized by different levels of resistance to the lethal effect of the oxidant does not correlate with resistance to DNA SSB-induction; d) a much larger accumulation of DNA SSBs can be observed following treatment with H2O2 at 4 degrees C, as compared to 37 degrees C, although the opposite is true in terms of cytotoxicity. In the presence of micromolar levels of L-Histidine, H2O2 also induces DNA double strand breaks (DSBs), a type of lesion which we suggest may mediate the lethal event. This conclusion finds experimental support in the following observations: a) DNA DSBs are generated at survival-range concentrations, and a linear correlation exists between the level of this lesion and cytotoxicity; b) this correlation curve overlaps with the curves generated under similar experimental conditions using different cell lines with different sensitivity to the oxidant alone, or different clones derived from the same cell line, some of which showed a high degree of resistance to H2O2. Finally, the formation of DNA DSBs appears to enhance both apoptotic and necrotic cell death.

Animals↗