Effect of Thickness and Translucency on Color Change and Masking Ability of Ceramic Materials used for Laminate Veneers.

This study evaluated the effect of the thickness and translucency of lithium disilicatebased glass ceramics on resin composite substrates on color change and masking effect. Laminate veneers were fabricated using IPS e.max CAD (A1) blocks with two different light transmittance values (High translucent [HT], Low translucent [LT]). Slices of two different thicknesses (0.3 mm, 0.5 mm) were obtained (n=10) and laminate veneers were cemented on the resin composite substrates of two different shades (A2, A3.5). The color change (ΔE values) was evaluated with the CIELab color system using a spectrophotometer, while the masking effect was calculated. The data were analyzed using independent-samples t-test and two-way analysis of variance. The ceramic thickness and translucency had a significant effect on final color and masking. When HT was used, and the laminate veneer thickness decreased (0.3 mm), the masking effect in ΔE values were lower (p⟨0.05). The ΔE values (⟩3.7) were clinically unacceptable. With the increase in thickness, translucency of porcelain laminate veneers decreases showing better color masking ability. Veneer thickness seems to be more effective on the restoration's masking ability than the shade of the substrate and translucency. Cinically, in case a 0.5-mm or thinner laminate veneer is planned, tooth color, resin cement and ceramic type should be considered.

Epithelioid trophoblastic tumor (ETT) initially interpreted as cervical cancer.

The epithelioid trophoblastic tumor (ETT) is a rare form of trophoblastic disease and shows a wide spectrum of differential diagnoses and clinical behavior. A 53-year-old woman presented with ETT presumably originated in spontaneous delivery of 25 years ago and was initially diagnosed as cervical cancer on cervical punch biopsy followed by radical hysterectomy. The uterus showed a small tumor restricted to the cavum with no cervical infiltration, resembling ETT in histologic and immunohistochemical features. The difficulties and clues in distinguishing ETT from nontrophoblastic lesions are discussed.

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450.

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.

Hemadsorption expressed by cloned H genes from subacute sclerosing panencephalitis (SSPE) viruses and their possible progenitor measles viruses isolated in Osaka, Japan.

Most subacute sclerosing panencephalitis (SSPE) viruses, including our Osaka-1, -2, and -3 strains isolated in Osaka, have shown negative hemadsorption (HAD) by African green monkey red blood cells. This property has been thought to be characteristic of SSPE virus as compared to the positive reaction of the standard Edmonston strain of measles virus (MV). However, this assumption has become quite obscure because MV mutates frequently at the genetic level during its multiplication and also because recent field strains isolated by lymphoblastoid cell lines have shown negative HAD. To investigate the above issue, the nucleotide sequences of the hemagglutinin (H) genes from SSPE virus Osaka-1, -2, or -3 strains were compared to those of various MV field strains isolated in Osaka by Vero cells. The H gene sequences of three SSPE strains were relatively conserved without such biased hypermutation as had been observed in the matrix (M) gene of three SSPE strains. However, this analysis of the H gene sequence of the SSPE viruses enabled us to deduce possible progenitor MVs, which are in agreement with the deduction from the M gene analysis we reported previously. The HAD of Vero cells transfected with the cloned H cDNAs from the SSPE strains and their progenitors suggested that negative HAD of the SSPE viruses has been maintained as one of original properties of the progenitor MVs rather than having been acquired as an altered one during long-term persistent infection in the brains of patients with SSPE.

Major change in the predominant type of "Norwalk-like viruses" in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, between April 1996 and March 1999.

In Osaka City, Japan, between April 1996 and March 1999, a total of 350 fecal specimens from 64 outbreaks of acute nonbacterial gastroenteritis were examined to investigate infection by "Norwalk-like viruses" (NLVs). By reverse transcription (RT)-PCR, 182 samples (52.0%) from 47 outbreaks (73.4%) were NLV positive. During those three years, the incidence of NLV-associated outbreaks showed seasonality, being higher during January to March (winter to early spring). The ingestion of contaminated oysters was the most common transmission mode (42.6%). The amplicons of the 47 outbreak strains that were NLV positive by RT-PCR were tested using Southern hybridization with four probe sets (Ando et al., J. Clin. Microbiol. 33:64-71, 1995). Forty of the outbreak strains were classified as 4 probe 1-A (P1-A) strains, 6 P1-B strains, 10 P2-A strains, 17 P2-B strains, and 3 untypeable strains, and the other 7 outbreaks were determined to be mixed-probe-type strains. Probe typing and partial sequence analysis of the outbreak strains indicated that a predominant probe type of NLVs in Osaka City had drastically changed; P2-B strains (77.8%) with multiple genetic clusters were observed during the 1996-97 season, the P2-A common strain (81.3%) related to the Toronto virus cluster was observed during the 1997-98 season, and P1-B strains (75.0%) with a genetic similarity were observed during the 1998-99 season. For the three untypeable outbreak strains (96065, 97024, and 98026), the 98026 outbreak strain had Southampton virus (SOV)-like sequences, and each of the other outbreak strains had a unique 81-nucleotide sequence. Newly designed probes (SOV probe for the 98026 outbreak strain and the 96065 probe for the 96065 and 97024 outbreak strains) were hybridized with relative strains and without other probe type strains. The prevalent NLV probe types in Osaka City during those three years were classified in six phylogenetic groups: P1-A, P1-B, P2-A, P2-B, SOV, and 96065 probe types.

Cell surface expression of immature H glycoprotein in measles virus-infected cells.

Two forms of hemagglutinin (H) protein, one with an apparent molecular mass of 78 kDa (78K H protein) and the other with that of 74 kDa (74K H protein), are present in cells infected with measles virus (MV). We previously observed that only the mature 78K H protein, a completely glycosylated form of the 74K H protein, was expressed on the cell surface of the infected cells. In the present study, we detected transient expression of the 74K H protein on the cell surface of infected cells by pulse-chase studies, although the level of this expression was much lower than that of the 78K H protein. On the cell surface the 74K H protein was present as dimers and sensitive to endo-beta-N-acetylglucosaminidase H digestion. Treatment with brefeldin A, which blocks the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus, inhibited the cell surface expression of the 78K H protein, but not that of the 74K H protein. These data suggest that a part of the MV 74K H proteins could be transported directly to the cell surface - probably via an alternative pathway - without processing to the complex form in the Golgi apparatus.

Different transcriptional expression of the matrix gene of the two sibling viruses of the subacute sclerosing panencephalitis virus (Osaka-2 strain) isolated from a biopsy specimen of patient brain.

Two sibling viruses of the subacute sclerosing panencephalitis (SSPE) virus Osaka-2 strain were isolated from a small biopsy specimen of the brain of an SSPE patient just before intraventricular interferon treatment by cocultivation with two different cell lines, Vero cells or B95a cells (Ogura et al, 1997). Both the virus-infected cells were found to be indistinguishable from each other in defective production of cell-free virus and in defective expression of the matrix (M) protein. The sequence analysis of the M genes predicted that they were translatable due to a lack of alteration of the translational start and stop codons for the proteins. A different pattern of the M monocistronic transcripts, however, was observed in a Northern blot analysis of the infected cells. This different pattern was confirmed further by a primer extension analysis. The undetectable expressions of the M proteins in the sibling virus-infected cells are most probably different in their molecular mechanisms. All these results indicate the possibility that the two different, replicable variants existed at Jabbour stage III of the disease's progression in a very small portion of the brain, where no lesion had yet been recognized by a magnetic resonance imaging.

Nucleotide sequences of the matrix protein gene of subacute sclerosing panencephalitis viruses compared with local contemporary isolates from patients with acute measles.

Measles viruses isolated from brain cells of patients with subacute sclerosing panencephalitis (SSPE) have numerous mutations, especially in the matrix protein (M) gene. To find whether the M genes of these SSPE viruses were mutated randomly or in a pattern, we sequenced this gene from three strains of defective measles virus isolated in Osaka, Japan. We could deduce the sequence of the possible progenitor measles virus for each patient by comparison of the isolate with measles viruses prevailing at roughly the same time and place as the primary infection. Biased hypermutation affected the M genes of all three SSPE viruses, although the molecular mechanisms for the mutations might be various. Replacements of U with C in the plus strand accounted for 76% of all mutations in two of the strains, but in the other strain, replacements of A with G accounted for 52% of the mutations, and the U residues were unchanged.

The matrix gene expression of subacute sclerosing panencephalitis (SSPE) virus (Osaka-1 strain): a comparison of two sibling viruses isolated from different lobes of an SSPE brain.

The Fr/V and Oc/V sibling viruses of the Osaka-1 strain of the subacute sclerosing panencephalitis (SSPE) virus were defective in cell-free virus production. By radioimmunoprecipitation assay, the matrix (M) protein was not detected in cells persistently infected with the Osaka-1 strain. This undetectable expression was consistent with the selective reduction of antibody response to the M protein in the patient from whom the Osaka-1 strain was isolated. The sequence of the M gene, however, predicted that the protein could be synthesized because the translational start and stop codons for the protein were not altered. Northern blot hybridization demonstrated the selective defect of the monocistronic mRNAs for the M protein and the phosphoprotein (P) together with the dominant presence of the P-M bicistronic mRNA. This absence of the M mRNA was further confirmed by primer extension analysis. Therefore, the undetectable expression of the M protein in the infected cells was proved to be caused by a transcriptional defect. The two sibling viruses, isolated from remote portions of an SSPE brain, were indistinguishable in their viral characters, including the M gene sequences, which indicates the possibility of clonal expansion of the strain in the brain.

Efficient isolation of subacute sclerosing panencephalitis virus from patient brains by reference to magnetic resonance and computed tomographic images.

Subacute sclerosing panencephalitis virus has been isolated with difficulty from brains of infected patients. More strains are needed for the study of the pathogenesis of this virus. To make the isolation more efficient, we selected portions to be examined from the brains of three patients with reference to findings of repeated magnetic resonance and computed tomographic imaging. Three cell lines susceptible to measles virus field strains were used. In all three cases viruses were isolated most effectively from recent lesions and with Vero cells. Our results suggested that these imaging methods and Vero cells could be used for improvement in the efficiency of isolation of this virus from patient brains.

Delayed activation of altered fusion glycoprotein in a chronic measles virus variant that causes subacute sclerosing panencephalitis.

We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE), Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.

Delayed activation of altered fusion glycoprotein in a chronic measles virus variant that causes subacute sclerosing panencephalitis.

We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE). Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.

Different antibody response to a neutralizing epitope of human cytomegalovirus glycoprotein B among seropositive individuals.

The amino-terminal portion of human cytomegalovirus glycoprotein B (HCMV-gB) was expressed as a fusion protein to analyze the neutralizing epitope recognized by human monoclonal antibody C23 and the humoral immune response to this epitope. The linear neutralizing epitope was further localized to the peptide within 17 amino acids (position 68-84) which were conserved between two HCMV laboratory strains. Ten out of 17 HCMV-seropositive human sera contained the antibody against this epitope. Although seven sera were negative for reacting with the fusion protein, the viruses isolated from the same patients retained the epitope. The immunogenicity of the epitope and the possible application of C23 human monoclonal antibody for passive immunization against HCMV infections are discussed.

Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding.

We have developed an in vitro nucleocapsid-binding assay for studying the function of the matrix (M) protein of measles virus (MV) (A. Hirano, A. H. Wang, A. F. Gombart, and T. C. Wong, Proc. Natl. Acad. Sci. USA, 89:8745-8749, 1992). In this communication we show that the M proteins of three MV strains that cause acute infection (Nagahata, Edmonston, and YN) bind efficiently to the viral nucleocapsids whereas the M proteins of four MV strains isolated from patients with subacute sclerosing panencephalitis (SSPE) (Biken, IP-3, Niigata, and Yamagata) fail to bind to the viral nucleocapsids. MV Biken (an SSPE-related virus) produces variant M sequences which encode two antigenically distinct forms of M protein. A serine-versus-leucine difference is responsible for the antigenic variation. MV IP-3 (an SSPE-related virus) also produces variant M sequences, some of which have been postulated to encode a functional M protein responsible for the production of an infectious revertant virus. However, the variant M proteins of Biken and IP-3 strains show no nucleocapsid-binding activity. These results demonstrate that the nucleocapsid-binding function is conserved in the M proteins of MV strains that cause acute infection and that the M proteins of MV strains that cause SSPE exhibit a common defect in this function. Analysis of chimeric M proteins indicates that mutations in the amino-terminal, carboxy-proximal, or carboxy-terminal region of the M protein all abrogate nucleocapsid binding, suggesting that the M protein conformation is important for interaction with the viral nucleocapsid.

Cytomegalovirus genomes demonstrated by polymerase chain reaction in synovial fluid from rheumatoid arthritis patients.

Synovial fluid from 20 patients with rheumatoid arthritis was analyzed to detect human cytomegalovirus (CMV) genomic material using polymerase chain reaction. Of 20 samples tested, 9 were positive for CMV by either ethidium bromide staining or Southern blotting. In contrast, CMV was detected in only 2 of 18 control patients with osteoarthritis. These results suggest an etiologic relationship between CMV and rheumatoid arthritis.

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.

Altered translation of the matrix genes in Niigata and Yamagata neurovirulent measles virus strains.

Niigata and Yamagata strains measles virus were isolated from subacute sclerosing panencephalitis patients. These viruses were defective in virion production and expression of matrix (M) protein. The Niigata M protein-coding frame was interrupted by an in-frame termination codon, whereas the Yamagata M gene lacked the normal translational initiation codon. These mutations prevented translation of a normal M protein. However, RNA derived from the cloned Niigata and Yamagata M genes was translatable in vitro into low levels of aberrant proteins that reacted with M-specific antiserum. These proteins were also translated from poly(A)+ RNA from cells infected by Niigata and Yamagata virus strains. The aberrant M protein of Niigata virus was initiated at a downstream AUG codon created by a second mutation. The Yamagata M gene produced two aberrant proteins: one initiated mainly in vitro at an ACG codon, and a second species initiated at a downstream site both in vitro and in vivo. These results define the abnormal translational functions of the Niigata and Yamagata M genes, and further implicate the involvement of M protein defects in chronic central nervous system infections by measles virus.

Malignant lymphoma of lymphoplasmacytic/lymphoplasmacytoid and immunoblastic types.

Sixty-four patients with extranodal non-Hodgkin's lymphomas of lymphoplasmacytic/lymphoplasmacytoid (LP) and immunoblastic (IBS) types were reviewed. The criteria distinguishing the latter from the former was a presence of basophilic and prominent nucleoli in a vast majority of the tumor cells. The numbers of LP and IBS types were 34 and 30, respectively. Intervals from appearance of tumors to initial treatment in LP and IBS were 15.7 +/- 25.3 and 6.0 +/- 12.3 months, respectively (p less than 0.1). Histologically LP had a low mitotic count (p less than 0.001) and contained a larger number of mast cells (p less than 0.05) than IBS. Proliferating cells in 4 patients with LP type had nuclei with a slightly irregular indentation similar to those of intermediate lymphocytic lymphoma. Follow-up study revealed the LP type to have a much more favorable prognosis than the IBS type (p less than 0.001) though 3 patients with LP showed unfavorable prognosis. Tumor cells in these tumors had a small to medium-sized, often convoluted nucleus. The present results showed that the distinction of LP and IBS by their morphology of nucleoli was prognostically meaningful. Furthermore it is suggested that tumors of the LP type are heterogeneous.

Generalized and localized biased hypermutation affecting the matrix gene of a measles virus strain that causes subacute sclerosing panencephalitis.

The matrix (M) genes of Yamagata-1 strain subacute sclerosing panencephalitis virus passaged in African green monkey kidney cells and human neuroblastoma cells displayed strikingly nonrandom sequence divergence. The genes of both substrains shared a large number of uridine (U) to cytidine (C) transitions, but the latter contained numerous additional U to C changes in a localized region. Over 90% of the additional mutations were identical to the hypermutated nucleotides in the M gene found in a measles inclusion body encephalitis case. The nonrandom nature, the apparent host dependency, and the abrupt boundaries of these mutations suggest that these mutations might be caused by an extrinsic biased mutational activity rather than intrinsic polymerase errors. This mutational activity might account for the extraordinarily high C to U ratios in the non-protein-coding regions of both the M and fusion genes of wild-type measles virus.

Roles of prepubertal androgen, estrogen or androgen plus prolactin on androgen-induced proliferative response of seminal vesicles in adult mice.

Male mice castrated on day 0 after birth were pretreated daily with testosterone propionate (TP, 4 micrograms/g body weight), 17 beta-estradiol (E2, 0.2 micrograms/g body weight) or vehicle for 21 days starting from day 20. In another experiment, male mice were castrated on day 25; two pituitaries from 60-day-old females were immediately grafted under the capsule of the left kidney in one group. The castrated mice with or without grafts were pretreated daily with TP (4 or 20 micrograms/g body weight) for 36 days starting from day 25, and the left kidney was removed on day 60. Daily TP injections (4 micrograms/g body weight) were started again at 30 days after the end of pretreatments to examine androgen-induced proliferation, and incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was used as an index of proliferation. In the neonatally castrated mice, both TP and E2 pretreatments given during the prepubertal period significantly increased seminal vesicle weight even long after the end of the pretreatments. However, androgen-induced proliferative response found in the neonatally castrated adult mice (poor response; long duration with a low peak) was changed to that found in mice castrated at adulthood (good response; short duration with a high peak) by the TP pretreatment only but not at all by the E2 pretreatment. In the mice castrated on day 25, a pharmacological dose of TP or TP plus hyperprolactin could not enhance or change the adult castration type of androgen-induced proliferation induced by physiological prepubertal androgens, although both treatments significantly enhanced the prepubertal growth of the seminal vesicles.