Location of main occluding areas and masticatory ability in patients with implant-supported prostheses.

BACKGROUND: The purpose of this study was to locate the main occluding area when the reduced posterior occlusal support was treated with an implant-supported prosthesis and to evaluate the subsequent improvement in the masticatory ability as compared with removable partial dentures. METHODS: Twenty-six patients with implant prostheses and 24 patients with removable partial dentures were recruited for this study. All patients had partially lost their posterior occlusal support. The first molar region in any quadrant was always included in the prosthetic region. Each subject was instructed to clench a piece of temporary stopping as a test food on the occluding area that was preferably used during mastication. The main occluding area was judged by locating the tooth on which the temporary stopping rested during clenching. Subjective masticatory ability was self-assessed by means of a questionnaire. RESULTS: The main occluding area of the subjects in the implant group was located more posterior compared with the removable partial denture group. The level of masticatory ability in the implant group was the same as that in the control group. CONCLUSIONS: The location of the main occluding area and the masticatory ability of the subjects with implants were equivalent to those with healthy natural dentition.

Noncovalent interaction of MNSFbeta, a ubiquitin-like protein, with histone 2A.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSFbeta, an isoform of the MNSF, has been isolated and characterized. MNSFbeta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Most recently, we observed that Ubi-L covalently conjugates to Bcl-G, a novel pro-apoptotic protein. In this study, we observed that Ubi-L noncovalently and specifically binds to histone 2A. The maximum binding was observed at a molar ratio equal to 1 for GST-Ubi-L and 2 for histone 2A. Ubi-L formed complex with histone 2A in the presence of 1% Triton X-100. Free Ubi-L was detected in nuclei from unstimulated murine helper T cell line, D10. The increased amounts of free Ubi-L and some Ubi-L adducts were observed in nuclei from mitogen-activated D10 cells. Interestingly, two Ubi-L adducts were unique to the chromatin fraction of nuclei from the activated D10 cells.

[A clinical study on patients detected Pasteurella multocida from sputum].

We reported ten cases, (four female and six male), whose sputum cultures positive for Pasteurella multocida from 1990 to 2000. In the past eleven years increasing numbers of cases have appeared in our hospital. The majority of the cases with P. multocida possessed some underlying pulmonary diseases (seven cases, 70%), inactive lung tuberculosis or bronchiectasis. There were compromised hosts such as high ages person, steroids dependent person and diabetes mellitus patients. P. multocida was almost susceptible to antibioticus (penicillin and cephalosporins), although some erythromycin resistant strains were identified. The cats' oral cavities in our two cases were cultured and P. multocida were isolated. In our survey the prevalence of this organism is as high as 85% in cats. Our data suggests that patients who are in the high infection risk category are easily infected to P. multocida.

Nitric oxide regulates actin reorganization through cGMP and Ca(2+)/calmodulin in RAW 264.7 cells.

Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (+/-)-(E)-2-[(E)-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNgamma was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or the arginine analogue N(G)-monomethyl-L-arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca(2+)/calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.

Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-gamma-treated RAW 264.7 cells.

The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-gamma-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-gamma-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-gamma-treated RAW 264.7 cells. PMA together with IFN-gamma increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-gamma-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-kappa B and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-gamma in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-kappa B. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-gamma-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-gamma-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.

Protein tyrosine phosphorylation induced by ubiquitin-like polypeptide in murine T helper clone type 2.

Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L shows an antigen-nonspecific immunosuppressive action on various target cells including murine T helper type 2 clone, D10 cells. Most recently, we have characterized the biochemical nature of the receptor for Ubi-L. In this study, we observed that Ubi-L receptor ligation rapidly and transiently stimulated tyrosine phosphorylation of 65- and 31-kDa proteins in concanavalin A-activated D10 cells. The addition of neutralizing antibody to Ubi-L receptor inhibited the protein tyrosine phosphorylations and the Ubi-L-mediated suppression of IL-4 production by D10 cells. Genistein, a tyrosine kinase inhibitor, also reduced the induction of these protein tyrosine phosphorylations. IFNgamma, which is also known to inhibit the proliferative response of D10 cells, showed a synergistic effect with Ubi-L. Interestingly, IFNgamma enhanced the Ubi-L-induced tyrosine phosphorylation of the 31-kDa protein. These results suggest that tyrosine phosphorylation may be a key step in the initiation of the Ubi-L receptor-mediated transmembrane signaling.

Biochemical analysis of the receptor for ubiquitin-like polypeptide.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-beta, an isoform of the MNSF, has been isolated and characterized. MNSF-beta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to unstimulated D.10 cells. Detergent-extracted membrane proteins were applied to an immobilized Ubi-L column. SDS-polyacrylamide gel electrophoresis of eluted fraction revealed a band of Mr = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of Mr = 90,000 was visualized by immunoprecipitation of 125I-Ubi-L cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide) antibody, indicative of the association of this protein with the Ubi-L receptor complex. Amino acid sequence analysis of the 82-kDa polypeptide revealed that the Ubi-L receptor may be a member of a cytokine receptor family.

Synergistic effect of ubiquitin on lipopolysaccharide-induced TNF-alpha production in murine macrophage cell line RAW 264.7 cells.

Ubiquitin synergistically augmented the production of tumor necrosis factor alpha (TNF-alpha) in the presence of lipopolysaccharide (LPS) in murine macrophage cell line RAW 264.7. To investigate the mechanism of this augmentation, we analyzed the effect of ubiquitin during TNF-alpha mRNA synthesis and degradation, and TNF-alpha degradation on RAW 264.7 cells stimulated by LPS. It is found that ubiquitin augmented TNF-alpha mRNA synthesis. Ubiquitin did not affect the degradation of TNF-alpha mRNA and TNF-alpha. In the presence of LPS, extracellular accumulation of TNF-alpha by ubiquitin was twice than those by LPS, but intracellular accumulation of TNF-alpha produced by ubiquitin with LPS or by LPS had no difference. These data indicate that ubiquitin might induce TNF-alpha accumulation mainly by up-regulation of the TNF-alpha gene transcription. Although extracellular functions of ubiquitin remain largely unknown, we postulate that ubiquitin might be involved in the modulatory mechanisms of immune response.

Ubiquitin-like polypeptide inhibits the proliferative response of T cells in vivo.

The monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Recently, we demonstrated that the recombinant form of the ubiquitin-like segment (rUbi-L) of MNSFbeta, a 15.6 kDa-protein consisting of a polypeptide with 36% homology with ubiquitin fused to the ribosomal protein S30, presented an antigen-nonspecific immunoregulatory action in a manner similar to native MNSF. Although this cytokine has been characterized in vitro, little is known about its effects in vivo. Thus, we investigated whether rUbi-L shows a suppressor activity in vivo. The proliferative response of Con A (5 microg/ml)-stimulated splenocytes of mice treated with rUbi-L (500 ng/body) was notably decreased in a dose-dependent manner (max. 57+/-20%). In contrast, administration of high dose ubiquitin (50 microg/body) showed a little, but significant, effect (30+/-7%). Interestingly, concomitant addition of ubiquitin inhibited Ubi-L-induced suppression. Mice injected with rUbi-L without gelatin did not show any suppressive effect. NA4 (1microg/body), a neutralizing monoclonal antibody against rUbi-L, abolished the Ubi-L-mediated suppression. Therefore, ubiquitin-like polypeptide may be implicated in the immune responses in vivo.

Cloning of the gene gob-4, which is expressed in intestinal goblet cells in mice.

We isolated the novel cDNA gob-4, which was shown to be expressed in intestinal goblet cells. The deduced amino acid sequence is similar to the gene coding for the Xenopus laevis cement gland-specific XAG-2. These sequence and expression data suggest this gene may be involved in the secretory function.

Cloning and identification of the gene gob-5, which is expressed in intestinal goblet cells in mice.

By using the large-scale in situ hybridization system (Analytical Biochemistry (1997) 254, 23-30), we isolated the cDNA gob-5, which is expressed in the intestinal goblet cells in mice. The transcript was also found to be abundantly expressed in small intestine, colon, stomach, and uterus and slightly expressed in trachea tissue. The gob-5 cDNA was shown to be 3 kb in size. After screening digestive tract tissues using our in situ hybridization method we demonstrate that gob-5 is expressed in mucous cells. The deduced amino acid sequence is similar to the gene which encodes the epithelial chloride channel in the bovine trachea.

Diagnosis of gastric cancer up to three years after negative upper gastrointestinal endoscopy.

BACKGROUND AND STUDY AIMS: The degree of accuracy of gastroscopy for the detection of gastric cancer is poorly understood. The aim of this retrospective study was to determine the accuracy of gastroscopy by using cancer registry records. PATIENTS AND METHODS: Gastroscopic examinations (n = 37094) conducted between 1984 and 1989 were studied by linking them with hospital-based and population-based (Fukui Prefecture, Japan) cancer registry records between 1984 and 1992. False-negative gastroscopies that had been carried out within the three years preceding the diagnosis of gastric cancer were identified. RESULTS: The numbers of true-positive, false-positive, and false-negative examinations carried out were 659, six and 155, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value were 81.0%, 100.0%, 99.1%, and 99.6%, respectively. The overall diagnostic accuracy of gastroscopy was 99.6%. There was little difference in sensitivity results between the patient groups with regard to reason for referral, type of endoscope used, experience of endoscopist, or location of gastric cancer. The percentage of tumours representing early gastric cancer, identified after false-negative gastroscopy, was lower for those situated in the cardia or gastric body than for those in the angular notch or the antrum. CONCLUSIONS: The accuracy of gastroscopy in the detection of gastric cancer is satisfactory, but false-negative results are sometimes obtained. We emphasize the importance of repeated endoscopic examination for the detection of gastric cancer.

Cloning of the novel gene intelectin, which is expressed in intestinal paneth cells in mice.

Using a large-scale in situ hybridization screening method, we isolated the cDNA gene intelectin, whose mRNA is expressed in small intestinal paneth cells in mice. Northern blot analysis revealed that the mRNA corresponding to the cDNA was 1.2 kp in length, and expression was specific to the tissue in the small intestine. We termed this gene intelectin because the deduced amino acid sequence is similar to the previously cloned oocyte lectin gene of Xenopus laevis. The function of intelectin may be involved in the defence of microorganisms.

TCR-alpha chain-like molecule is involved in the mechanism of antigen-non-specific suppression of a ubiquitin-like protein.

Although existence of suppressor T cells is a controversial issue in cellular immunology, several lines of evidence indicate that T-cell-receptor alpha-chain (TCR-alpha) is a critical component of suppressor factors produced by these cells. Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by murine T-cell hybridoma, possesses pleiotrophic antigen-non-specific suppressive functions. Recently, we have shown that the 70,000-MW MNSF comprises an 8000-MW ubiquitin-like polypeptide and other subunit(s). Here we report that the 8000-MW ubiquitin homologue is associated with an intracellular TCR-alpha (but not TCR-beta)-like molecule and released from the cells. The affinity eluates obtained from the culture supernatants of E17 cells and concanavalin A (Con A)-activated splenocytes with anti-TCR-alpha monoclonal antibody (mAb) showed an antigen-non-specific, major histocompatibility complex (MHC)-non-restricted suppression. Immunoblot analysis demonstrated that anti-TCR-alpha, but not anti-TCR-beta, mAb recognizes native 70,000-MW MNSF. In addition, we found the dissociation of the 8000-MW polypeptide from the 62,000-MW TCR-alpha cross-reactive protein by hydrolase which cleaves isopeptide bonds. Thus the covalent attachment of ubiquitin-like protein(s) may be involved in the underlying mechanism of suppressor T-cells and TCR-alpha-like molecule(s) might be a main link between antigen-specific and non-specific suppression.

cFKBP/SMAP; a novel molecule involved in the regulation of smooth muscle differentiation.

During embryogenesis, smooth muscle cells of the gut differentiate from mesenchymal cells derived from splanchnic mesoderm. We have isolated a gene involved in the differentiation of smooth muscle cells in the gut using differential display between the chicken proventriculus in which the smooth muscle layer develops poorly and the gizzard in which smooth muscles develop abundantly. The protein encoded by this gene showed highest similarity to mouse FK506 binding protein, FKBP65, and from the function of this protein it was designated chicken FKBP/smooth muscle activating protein (cFKBP/SMAP). cFKBP/SMAP was first expressed in smooth muscle precursor cells of the gut and, after smooth muscles differentiate, expression was restricted to smooth muscle cells. In organ culture of the gizzard, the differentiation of smooth muscle cells was inhibited by the addition of FK506, the inhibitor of FKBPs. Moreover, overexpression of cFKBP/SMAP in lung and gizzard mesenchymal cells induced smooth muscle differentiation. In addition, cFKBP/SMAP-induced smooth muscle differentiation was inhibited by FK506. We postulate therefore that cFKBP/SMAP plays a crucial role in smooth muscle differentiation in the gut and provides a powerful tool to study smooth muscle differentiation mechanisms, which have been poorly analyzed so far.

Production and characterization of a monoclonal antibody specific for ubiquitin-like polypeptide responsible for nonspecific immune suppression.

Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine product of a murine T cell hybridoma that inhibits the immune response in an antigen nonspecific manner. Recently, we found that a novel ubiquitin-like protein (Ubi-L), a subunit of MNSF, is responsible for its biological activity. We developed a monoclonal antibody with specific activity against Ubi-L. Inhibition experiments showed that this mAb, termed NA4, preferentially recognizes Ubi-L but not irrelevant proteins such as ubiquitin. With the use of NA4, we established an ELISA method for the quantitation of Ubi-L. By this ELISA system, approximately 40 ng/ml of MNSF was detected in the culture supernatants of concanavalin A (Con A)- or interferon gamma (IFN gamma)-activated splenocytes, whereas MNSF in the supernatant of IFN alpha- and IFN beta-stimulated splenocytes was nil. In addition, NA4 could abrogate the action of Ubi-L. Thus NA4 was confirmed to be a pertinent tool for elucidation of the underlying mechanism of action of MNSF.

Ubiquitin-like polypeptide conjugates to acceptor proteins in concanavalin A- and interferon gamma-stimulated T-cells.

Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by a murine T-cell hybridoma, possesses pleiotrophic non-specific suppressive functions. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% homology with ubiquitin and ribosomal protein S30. The ubiquitin-like segment of MNSFbeta (Ubi-L) is an 8 kDa polypeptide with MNSF-like activity. Since the amino acids critical for the ubiquitination process are conserved in Ubi-L, we examined whether Ubi-L may conjugate with intracellular proteins in a manner similar to the ubiquitin system. Rabbit polyclonal antibodies specific for Ubi-L detected the induction of Ubi-L conjugations, including 33.5 kDa and 70 kDa molecules in concanavalin A (Con A)-stimulated T-cells, but not in lipopolysaccharide-stimulated B-cells and macrophages. High-molecular-mass conjugates were consistently present in pan-T-cells. However, free Ubi-L could not be observed in all the cells tested. Con A-activated CD8+ T-cells, but not CD4+ T-cells, induced the 70 kDa Ubi-L adduct, which was recognized by an anti-MNSF monoclonal antibody. Treatment of CD8+ T-cells with interferon (IFN) gamma also caused the expression of the 70 kDa Ubi-L adduct, whereas the responses to IFNalpha and IFNbeta were nil. Antigen- and Con A- stimulated D.10 G4.1, a murine T helper clone type 2, induced the 33.5 kDa, but not the 70 kDa, adduct. These results suggest a role for Ubi-L conjugation in the regulation of T-cell activation.

Conjugation of ubiquitin-like polypeptide to intracellular acceptor proteins.

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine hybridoma, was originally found to inhibit the generation of LPS-induced immunoglobulin secreting cells. MNSF comprises of MNSF beta, an isoform of MNSF, and the other isoform, MNSF alpha. Ubiquitin-like segment (Ubi-L) of MNSF beta shows MNSF-like activity. Ubi-L (7.8 kDa) has 36% homology with 8.5 kDa ubiquitin. GST-Ubi-L was labeled with 125I by the chloramine T method and tested for its conjugation to acceptor in splenocyte lysates. 125I-GST-Ubi-L conjugation on SDS-PAGE showed heterogeneous bands including 95 kDa GST-Ubi-L conjugation in the splenocyte, but not reticulocyte lysates. The Ubi-L adduct appeared to be MNSF-related molecule because anti-MNSF monoclonal antibody (mAb) recognized the 95 kDa band. The pattern of the conjugations was different from that seen in ubiquitination. Unlabeled GST-Ubi-L inhibited the conjugations, while ubiquitin did not. alpha-Lactalbumin, one of the target proteins for ubiquitination, failed to conjugate to GST-Ubi-L. In addition, covalent conjugation of ubiquitin to reticulocyte lysates was also interfered by GST-Ubi-L. These results suggest that Ubi-L may conjugate to acceptor proteins in a similar, but not in the same way as ubiquitination, and might play an important role in lymphoid cells.

Large-scale immunohistological staining using polyethylene glycol-embedded sections mounted on 96-well plates for monoclonal antibody screening.

We have developed a rapid, large-scale immunohistological staining method for monoclonal antibody screening. Sections embedded in polyethylene glycol are mounted on 96-well plates and then subjected to immunological procedures using a 96-well format. With this method, we can prepare 10 plates (960 samples) in 1 day and then perform immunohistological procedures in 1 day by using an enzyme-linked immunosorbent assay-like 96-well format. Thus, we can avoid difficulties seen in conventional immunohistological methods that use sections mounted on glass slides. This method offers high-throughput screening of monoclonal antibodies. In addition, it has the potential of being automated.

[An autopsy case of chronic granulomatous disease diagnosed by biopsy].

This report concerns a male patient aged 25 years, diagnosed at the age of 12 years as suffering from chronic granulomatous disease. This patient had p47-phox deficiency. He was admitted to this hospital because of fever and dyspnea accompanied by right spontaneous pneumothorax. He failed to respond to medical treatment. He died from respiratory failure four months after admission. Autopsy demonstrated pigmented lipid histiocytes characteristic of CGD. These characteristic pigmented cells were distributed in the spleen, liver, lymph nodes and in the small intestine. As for the nature of the pigment, lipofuschin-like compound were identified. Granulomatous component was seen in the mucosa of the stomach obtained by operation. The presence and characteristic distribution of such pigmented macrophages in tissue in young adults may suggest the diagnosis of CGD.