Silicoating in resin-metal bonding: restorative implications for osseointegrated implant-bone unison.

Detailed background information leading to the development of a silicoating technique is presented with its advantages and disadvantages and its role in implant dentistry. The rationale is shown for coining a new acronym "osseointegrated Implant-Bone Unison" with an interest in biomechanical considerations for a prosthodontic restorative scenario. Several clinical cases are presented that involve silicoating and composite restorations.

Increase in brain nitric oxide synthase activity in daunorubicin-treated rats.

Anthracyclines such as daunorubicin are very effective anticancer agents. These drugs are known to cause side effects including cardiotoxicity. Anthracyclines are neurotoxic to laboratory animals. Nitric oxide is a novel and very important chemical messenger in the brain. However, at higher levels, nitric oxide causes well defined neurotoxicity. Therefore, we determined nitric oxide synthase activity in rat brain after daunorubicin treatment in an effort to explain the neurotoxicity produced by anthracyclines. Male Sprague-Dawley rats were treated with different subcutaneous doses of daunorubicin (0.1-4.0 mg/kg/week for five weeks) while control animals were injected with phosphate buffered saline. There was a significant increase (80%) of nitric oxide synthase activity in daunorubicin-treated animals as compared to controls. This activity was inhibited by N-monomethyl-L-arginine (NMMA), nitroarginine, N-6-aminohexyl-5-chloro-1-napthalene sulfonamide (W-7), a calmodulin antagonist, suggesting that the nitric oxide synthase activity is calmodulin dependent. Further, our in vitro studies demonstrated that daunorubicin interacted with calmodulin as measured by N-phenyl-1-napthylamine (NPN) fluorescence. These results indicate that daunorubicin increases nitric oxide synthase activity in rat brain which may increase the levels of nitric oxide. The increased levels of nitric oxide may cause neurotoxicity. Our results further indicate that daunorubicin interacts with calmodulin and enhances nitric oxide synthase activity which is dependent on calmodulin.

Characterization of primary human fibroblasts transformed by human papilloma virus type 16 and herpes simplex virus type 2 DNA sequences.

Human papilloma virus type 16 (HPV-16) and herpes simplex virus type 2 (HSV-2) are human viruses implicated in the development of cancer, in particular cervical cancer. The ability of HSV-2 and HPV-16 to transform early passage human cells was examined in this report. For these studies, gingival fibroblasts were utilized. One gingival cell strain was derived from a normal individual (N-16). The second cell strain was derived from hyperplastic gingival tissue of an epileptic individual (R-30) treated with phenytoin, an antiseizure drug. A common side effect of phenytoin is the induction of gingival overgrowth. R-30 cells contained a stable chromosomal translocation between chromosomes 8 and 18 and expressed higher steady state levels of c-myc. HPV-16 DNA efficiently immortalized R-30 cells but not N-16 cells. R-30 cells cotransfected with HPV-16, and HSV-2 viral DNAs were more aneuploid than R-30 cells transfected with HPV-16 DNA alone. Additionally, R-30 cells cotransfected with both viral DNAs grew better in soft agar than R-30 cells transfected with HPV-16 DNA alone. HSV-2 DNA was detected in transformed cells by polymerase chain reaction. These results suggested R-30 cells were immortalized more efficiently by HPV-16 and further imply that HPV-16 and HSV-2 DNA fragments can cooperate during multistep transformation.

Herpesviral deoxythymidine kinases contain a site analogous to the phosphoryl-binding arginine-rich region of porcine adenylate kinase; comparison of secondary structure predictions and conservation.

Twelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple alignment. Six highly conserved sites were observed. Site 1 corresponded to a glycine-rich loop that forms part of the ATP-binding pocket in porcine adenylate kinase (PAK), and site 5 corresponded to a region in PAK, located on one lobe of the cleft, that contains arginine residues that bind substrate phosphoryl groups. Site 3, consisting of the motif -DRH-, is thought to be involved in thymine/deoxythymidine recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2 and 6 have not been identified. Secondary structure predictions were made by the Garnier method and averaged for each position in the multiple alignment. The structure predicted for all six sites was typically a short flexible region (turn or coil) at or adjacent to the site, flanked by rigid structures (helix or sheet) on either side.

Equine herpes virus 1 and pseudorabies virus resistance to 2'-fluoropyrimidine analogs and to bromovinyldeoxyuridine: implications for dTMP kinase activity.

The 2'-fluoropyrimidine nucleoside analogs 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC). 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), and 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) showed higher in vitro activity against herpes simplex virus type 1 (HSV-1), than equine herpesvirus 1 (EHV-1) or pseudorabies virus (PRV). Comparison of the 50% plaque inhibitory doses for HSV-1 and its mutant MMdUr-20 in cell cultures with inhibition constants (Ki's) for the viral deoxythymidine kinases (dTKs) suggests that in the infected cell FMAU is phosphorylated by host enzymes. As compared to HSV-1, EHV-1 and PRV were more resistant to E-5-(2-bromovinyl-2'-deoxyuridine (BVdU) and to the 2'-fluoropyrimidine analogs, as are HSV-2 and the HSV-1 mutants MMdUr-20 and S1. Because the dTKs of the latter lack deoxythymidylate kinase (dTMPK) activity, there appears to be a correlation between resistance to these analogs and BVdU on the one hand, and lack of dTMPK activity on the other. We predict that EHV-1 and PRV dTKs will be shown to lack significant dTMPK activity.

Studies on interactions of dTK-HSV mutants with neurons in vitro.

Thymidine kinase negative (dTK-) mutants of herpes simplex virus type 1 (HSV-1) multiplied well in rat brain glioma cells. A proportion (less than 1%) of glioma cells survived the infection with HSV and were designated "survivor" glioma cells. Survivor cells of dTK- mutant virus infection ceased to produce infectious virus after two passages and were highly resistant to both HSV-1 and HSV-2 but not to vesicular stomatitis virus (VSV). Flow cytometric studies indicated morphological differences between parental and survivor glioma cells, and HSV-1 specific antigens as well as DNA were detected in the survivor glioma cells, but only in early passages. Sensitivity to superinfection with HSV appears to correlate to loss of HSV-specific viral DNA in the survivor glioma cells. Survivor glioma cells after several subcultures lost their ability to resist superinfecting HSV, reverted morphologically to the appearance of parental glioma cells and also lost significant amount of HSV-1 specific DNA. These transient survivor glioma cells became persistently infected-virus producer cells upon HSV infection.

Herpes simplex virus type 1 and neuronal cells--a special cell-virus interaction.

Rat brain glioma cells were semipermissive for herpes simplex virus (HSV) replication, because the growth of HSV was multiplicity-dependent in these cells. By using this property, we successfully isolated 'survivor' glioma cells following HSV infection at low multiplicity and without using any special treatment (such as UV irradiation) either of the cells or of the virus. Under the same conditions there were no survivor BHK or 3T3 cells, which suggests the uniqueness of the glioma cell-HSV interaction. The survivor cells ceased to produce infectious virus after two subcultures, but were highly resistant to superinfection for at least 20 subcultures. Parental cells were significantly more permissive for homologous virus growth than survivor cells. Interferon was apparently not induced in the survivor cells, because they were as susceptible as the parental cells to infection with vesicular stomatitis virus. The survivor cells produced HSV-specific antigens and contained HSV-specific DNA.

HSV1-specific thymidylate kinase activity in infected cells.

Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5-bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs.

Alterations in the recognition of nucleoside analogues as substrates by the deoxythymidine kinase of a 5-methoxymethyldeoxyuridine-resistant mutant of herpes simplex virus type 1.

Inhibition constants (Kis) were used as an estimate of the ability of various nucleoside analogues to be recognized as substrates by the deoxythymidine kinases (dTKs) of a 5-methoxymethyldeoxyuridine-resistant (MMdUr) mutant of herpes simplex virus type 1 (HSV-1) and its parent wild-type (wt). It was found that the Kis for the 5-position analogues MMdU, [E]-5-(2-bromovinyl)deoxyuridine, bromodeoxyuridine and iododeoxyuridine were increased approximately three-to fivefold, suggesting that they were poorer substrates for the MMdUr dTK than for the wt dTK. With the 2' analogues arabinosylthymine and 2' fluoro 5-methylarabinosyluracil, however, the Kis were increased to a much greater extent, 80- and 240-fold, respectively. These findings suggest that the resistance of the mutant MMdUr to these analogues may be due to a mutation(s) in the viral dTK that directly affects binding at the 2' recognition site and indirectly at the 5, while still allowing substantial activity with the natural substrate deoxythymidine.

Alterations in substrate specificity and physicochemical properties of deoxythymidine kinase of a drug-resistant herpes simplex virus type 1 mutant.

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).

Thymine salvage, mitochondria, and the evolution of the herpesviruses.

The salvage of thymine is an apparently ubiquitous feature of free-living lifeforms as well as of mitochondria, chloroplasts and most of the large DNA viruses. Assumptions and data are described which explain the evolution of thymine salvage in prokaryotes, animal cells, and large DNA viruses, in terms of deoxythymidine kinase and its relationship to mitochondria. Specifically, it is suggested that regulation of deoxythymidine kinase (by end-product inhibition) has evolved as a means of assuring a constant supply of thymine compounds for the mitochondria and that the degree to which this regulation is present in the deoxythymidine kinases of the various herpesviruses correlates with the degree of dependence of their replicative cycle on the continued health of the mitochondria of their host cells.

Temperature-induced structural stabilization of the encapsidated RNA of southern bean mosaic virus and its biologic significance.

Virions of southern bean mosaic virus (SBMV) show two distinct sensitivity patterns upon heating. Infectivity loss and capsid denaturation occur concurrently if virions are exposed at 50-55 degrees in 0.1 M sodium phosphate buffer, pH 7.5, or 0.1 M glycine-phosphate buffer, pH 9.0. Contrastingly, virions are inactivated without any detectable capsid damage at 65-70 degrees in 0.1 M sodium phosphate buffer, pH 6.0, 0.1 M Tris-HCI buffer, pH 7.5, or 0.1 M glycine-NaOH buffer, pH 9.0. Heat treatment causes no genomic degradation in these two situations; the divergent sensitivity of virions is due, apparently, to a differential thermal tolerance of the capsid protein to the buffer components and/or pH. SBMV-RNA isolated from virions inactivated at 65 degrees in 0.1 M Tris-HCl buffer, pH 7.5, possesses low infectivity (1-2%). Observations based upon sucrose gradient sedimentation, temperature: absorbance relationship, and sensitivity to ribonuclease T(1) suggest that such RNA is structurally more compact and stable relative to that of the RNA from the nonheated virions. Neither the capsid protein nor the genome protein plays a direct role in the temperature-induced structural stabilization of SBMV-RNA in situ. If treated with 8 M urea, 50% formamide or exposed at 55 degrees , the infectivity of RNA from the heat-inactivated SBMV is restored and is comparable to that of the RNA isolated from the nonheated virions. The observed mode of SBMV thermal inactivation, i.e., stabilization of RNA structure in situ, is unique among the viruses. Furthermore, these results suggest that the ability to initiate infection depends upon the secondary structure of the SBMV genome.

Suggestions for the classification and nomenclature of helical plant viruses.

Helical plant viruses are arranged into four families: Flexiviridae, Tobamoviridae, Hordeiviridae, and Tobraviridae. Further division of these families into genera and subgenera is shown. A family-specific, constant amount of RNA per unit virion length and a distinct radial location of RNA from the center of the helix are considered as basic criteria for establishment of these families.

Differentiation of legume carlaviruses based on their biochemical properties.

Nucleic acids of alfalfa latent virus (ALV), pea streak virus (PSV), and red clover vein mosaic virus (RCVMV) were hydrolyzed by pancreatic RNase but not by DNase. ALV and PSV each had one single-stranded RNA of 38.0 and 37.6 S, respectively, whereas two single-stranded RNAs of 39.1 and 12-15 S were found in the RCVMV preparations. The average apparent molecular weights obtained by sedimentation and electrophoresis of formalinized ALV-RNA, PSV-RNA, and the large RNA of RCVMV were 2.45, 2.55, and 2.59 x 10(6), respectively. The apparent molecular weight of the protein subunit of ALV was 27,000, whereas those of PSV and RCVMV were each 33,500, indicating that ALV is a distinct member of the carlavirus group. RCVMV and PSV are considered distinct viruses, since formalinized RCVMV-RNA, but not PSV-RNA, was resolved from formalinized tobacco mosaic virus RNA on gradients and since the capsid protein subunit of RCVMV resisted degradation on storage but that of PSV did not.