Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor ß (TGF-ß) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-ß, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-ß type I receptor, the cultures were supplemented with SB431542. Secreted TGF-ß was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-ß release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-ß type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

A Novel Diagnostic System for Infectious Diseases Using Solid-State Nanopore Devices.

Nanopore-based diagnostic systems are a promising tool for counting viruses in a specimen one by one. However, despite intensive R&D efforts, it remains difficult to recognize virus subtypes by nanopore devices. We thus propose a novel diagnostic system that combines a specialized virus recognition procedure with a nanopore detection procedure. This recognition procedure consists of three steps: 1) capture target viruses using specific probes for recognition; 2) release captured targets; and 3) detect released targets by nanopore. Proof-of-concept tests are conducted using avidin-modified fluorescent particles (as a model for viruses) and biotin-modified alkane thiol (as a model for probes). The avidin-modified particles are confirmed to be captured on electrode by biotin-modified probes and then, the particles are electrochemically released from the electrode. Consequently, the released particles are successfully detected by nanopore devices. Furthermore, the concept is also proved by using human influenza viruses (H1N1, A/PR/8/34) and sugar chain (6'-sialyllactose)-modified probes. This suggests that our concept is applicable to various infectious diseases by changing probes (ligands).

Some molecular properties of asparagine synthetase from rat liver.

Asparagine synthetase purified from rat liver reveals two species (slower migrating band I and faster migrating band II) when subjected to polyacrylamide gel electrophoresis under nondenaturing conditions (S. Hongo and T. Sato (1981) Anal. Biochem. 114, 163-166). We have investigated some molecular properties of these species. Elution of band I from the gel and re-electrophoresis showed that band I yielded band II similar to that of the initial run. Peptide maps by limited proteolysis were very similar and amino acid compositions were also alike in the two species. L-Lysine was identified as the sole NH2-terminal amino acid in both the species. By cross-linking experiments the enzyme was shown to be a dimeric protein. When the purified enzyme was subjected to isoelectric focusing the enzyme activity and protein focused at pH 6.0 in a single peak. These results demonstrate that rat liver asparagine synthetase is composed of two identical subunits. The enzyme, inactivated by storage at -20 degrees C for about 3 months, showed aggregated forms in polyacrylamide gel electrophoresis, and was reactivated markedly by the addition of dithiothreitol.

Purification and properties of asparagine synthetase from rat liver.

Asparagine synthetase (L-aspartate:ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation. DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4-7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110,000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl- were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by L-asparagine, nucleoside triphosphates except ATP, and sulfhydryl reagents. It has been observed that the properties of asparagine synthetase from rat liver are not so different from those of tumors such as Novikoff hepatoma and RADA 1.

Decrease in asparagine synthetase activity during cell differentiation of mouse and human leukemia cell lines.

The activity of asparagine synthetase decreased almost 50% during dexamethasone-induced mouse myeloid leukemia M1 cell differentiation. This enzyme activity also declined significantly during differentiation of the human myelogenous leukemic cell lines, HL-60 and U-937, induced by either macrophage culture supernatant or retinoic acid. The decline of asparagine synthetase activity closely paralleled the expression of various maturation markers, but could also be induced by serum starvation. These results suggest that asparagine synthetase or L-asparagine has some biological function in growth regulation of these leukemia cell lines.

Comparison of receptor-binding properties among influenza C virus isolates.

A total of 10 influenza C virus strains isolated recently in Yamagata City, Japan and shown to belong to the same lineage was compared for the ability to agglutinate chicken and mouse erythrocytes under various conditions. C/Yamagata/10/89 was unique in lacking the ability to agglutinate chicken erythrocytes at a temperature > or = 4 degrees C. This isolate also agglutinated native mouse erythrocytes only very inefficiently, although the high agglutination titer was obtained with the glutaraldehyde-fixed cells. Furthermore, it was found that C/Yamagata/4/88, unlike the other isolates, agglutinated erythrocytes from chickens to lower titers than those from mice, even when assayed at 0 degree C. Comparison of the deduced amino acid sequence of hemagglutinin-esterase among the 6 representative strains including two older isolates, C/Yamagata/26/81 and C/Nara/2/85, suggested that the failures of C/Yamagata/10/89 to agglutinate chicken erythrocytes at > or = 4 degrees C and unfixed mouse erythrocytes to high titers may be due to amino acid changes at residues 337 (Glu-->Lys) and 340 (Thr-->Tyr), respectively, and that a change at residue 347 (Leu-->Ser) may be responsible for the decreased ability of C/Yamagata/4/88 to agglutinate chicken erythrocytes.

Location of a linear epitope recognized by monoclonal antibody S16 on the hemagglutinin-esterase glycoprotein of influenza C virus.

We reported previously that monoclonal antibody S16, which had been raised against the hemagglutinin-esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 (AA/50) virus, recognizes a linear epitope present on the HE molecules of all influenza C viruses examined except for viruses belonging to a lineage represented by Aichi/1/81 (AI/81). Comparison of the deduced amino acid sequence of HE between viruses on the AI/81-related lineage and those on the others suggests that the epitope recognized by S16 is located in a region containing amino acid residue 403 and that a change from Glu to Lys at this position causes the loss of reactivity with the antibody. To prove it, the wild type (WT) HEs of AA/50 and AI/81 as well as their mutants with an amino acid substitution at residue 403 were expressed in CV-1 cells from the recombinant simian virus 40 (SV40) and tested for reactivity with S16 by immunoprecipitation. The results showed that the AA/50 virus WT and AI/81 virus mutant HEs (both having Glu at residue 403) were reactive with S16 whereas the AI/81 virus WT and AA/50 virus mutant HEs (both having Lys at residue 403) were not. Furthermore, we examined the reactivity of S16 with two synthetic peptides (corresponding to residues 397-409) that possess Glu and Lys at position 403, respectively, by enzyme-linked immunosorbent assays. The results demonstrated that the former peptide but not the latter was reactive with S16. These observations support strongly the notion described above. During this study it was also found that S16 cross-reacts with large T antigen of SV40.

Phosphorylation of influenza C virus CM2 protein.

Labeling of influenza C virus-infected HMV-II cells with [32P]orthophosphate showed that the CM2 protein is posttranslationally modified by phosphorylation. The unglycosylated form of CM2 synthesized in the presence of tunicamycin was found to be highly phosphorylated. This result, together with the finding that digestion of CM2 with peptide-N-glycosidase F failed to remove the 32P label from the glycosylated form of CM2, indicated that phosphorylation occurs in the polypeptide backbone and not in the oligosaccharide chain. Furthermore, phospho-amino acid analysis revealed that phosphorylation occurs exclusively on serine residues. Treatment of infected cells with brefeldin A resulted in a complete inhibition of phosphorylation, showing that phosphorylation of CM2 occurs after its migration from the endoplasmic reticulum to the Golgi apparatus. Phosphorylation of CM2 was also inhibited strongly, although not completely, by monensin treatment, suggesting that CM2 is phosphorylated predominantly after its movement from medial to trans Golgi cisternae. Finally, we found that the CM2 protein incorporated into the progeny virion is phosphorylated, which indicates that there is no strictly selective incorporation of the unphosphorylated form of CM2 into the virion.

Interspecies transmission of influenza C virus between humans and pigs.

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain.

Postnatal development, such as synapse refinement, is necessary for the establishment of a mature and functional central nervous system (CNS). Using differential display analysis, we identified a novel gene, termed Bdm1, that is more abundantly expressed in the adult brain than in the embryonic brain. The full-length Bdm1 cDNA is 2718 base pairs long and contains an open reading frame of 1059 base pairs encoding a 38-kDa protein. Northern blot analysis revealed that expression of Bdm1 mRNA in the brain was weak on embryonic days and increased in the early postnatal period. Bdm1 mRNA was significantly expressed in the brain and heart, but there was no or little expression in other tissues. During the differentiation of mouse carcinoma cells P19 to neuron-like cells by retinoic acid, Bdm1 mRNA was up-regulated almost parallel to neurofilament mRNA. Expression of Bdm1 mRNA was observed appreciably in PC12 cells after neuronal differentiation but not in the nonneural cell lines examined. In situ hybridization demonstrated that Bdm1 was expressed widely in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata. Taken together, these data suggest that Bdm1 gene plays a role in the early postnatal development and function of neuronal cells.

Plasma stromal cell-derived factor-1 during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization.

In this report, we examined plasma stromal cell-derived factor-1 levels in normal healthy donors for allogeneic peripheral blood stem cell transplantation (PBSCT) and in patients for autologous PBSCT using an enzyme-linked immunosorbent assay. The average level of plasma stromal cell-derived factor-1 was 2197 pg/ml before granulocyte colony-stimulating factor administration and 1899 pg/ml on day 4, demonstrating a significant decrease in the peripheral blood of healthy donors (P=0.0003). In patients for autologous PBSCT, a significant decrease of plasma stromal cell-derived factor-1 in the peripheral blood was also observed (P=0.0464). However, the physiologic gradient of stromal cell-derived factor-1 between peripheral blood and bone marrow was never inverted in normal healthy donors or in autologous PBSCT patients. Our results suggest that stromal cell-derived factor-1 may not be involved in the granulocyte colony-stimulating factor-induced release of CD34(+) cells to the peripheral blood. Further studies of a possible additive effect of granulocyte colony-stimulating factor and stromal cell-derived factor-1 are warranted.

Effect of dietary protein content on the activity of rat liver asparagine synthetase.

The activity of rat liver asparagine synthetase [EC 6.3.1.1]increased when animals maintained on 25% protein diet were placed on 15% or 6% protein diet. The enzyme activity level rose within one day, reached a maximum in 7 or 10 days after switching the diet and thereafter dropped gradually. During the purification of the enzyme from rats on 25% or 6% protein diet, the yield and increase of the specific activity were similar in the two groups. Combination of the liver extracts from two such groups demonstrated that the amount of endogeneous inhibitors of the enzyme did not change on replacing the diet. The elevation of the enzyme activity in rats fed 6% casein diet was suppressed by an injection of cycloheximide or actinomycin D. It is suggested that the change in the enzyme activity was due to alteration of the amount of the enzyme.

Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain.

Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells.

Expression of asparagine synthetase mRNA through asparagine independent signal transduction pathway that might involve protein kinase C in BALB3T3 cells.

Basal level of asparagine synthetase mRNA in BALB3T3 cells was elevated when the cells were shifted from medium containing a high concentration (3.3 mM) of asparagine to one lacking asparagine. We then studied whether the expression of asparagine synthetase mRNA is also mediated through other asparagine-independent signaling pathways. BALB3T3 cells grown to near confluence were quiesced by serum-starvation, and various agents were then added to the culture to examine the enzyme activity and mRNA level of asparagine synthetase. 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C (PKC), elevated dose and time dependently the level of asparagine synthetase mRNA even in Eagle's minimum essential medium with alpha modification (MEM alpha) that contains protein-constituting 20 amino acids and is supplemented with 3.3 mM asparagine. Staurosporine and H-7, PKC inhibitors, strongly blocked the fetal bovine serum-dependent accumulation of asparagine synthetase mRNA. TPA could also enhance the activity of asparagine synthetase within 24 h at concentrations of more than 10 nM. These results suggest that expression of asparagine synthetase gene can be induced both through a pathway that involves PKC and through a pathway the origin of which is a reduced concentration of asparagine in BALB3T3 cells.

Mechanism for splenocyte-mitogenesis induced in mice by water-extract of coniferous slash pine.

A water extract of wood chips of coniferous slash pine induced appreciable splenocyte-mitogenesis in healthy adult, aged, and also tumour-bearing mice. Mitogenesis was induced mainly in B-cell subset of splenocytes. The induction was not mediated by IL-2 or IFN gamma released from the activated helper T-cell. It may be worth noting that this extract could be included in a new type of immunomodulator for promotion of the host defense mechanism.

The atelectatic ear and its classification.

Atelectasis of the middle ear cavity occurs in advanced chronic otitis media with effusion. Atelectasis with effusion may progress to atelectasis without effusion, and further possible complications include adhesive otitis, ossicular disruption, cholesteatoma, and sensorineural hearing loss. A classification is presented of the various sequelae of otitis media with effusion under the basic concept of the atelectatic ear because most of the sequelae observed had developed through the atelectatic ear. This classification depends on localization of the main pathology, i.e. the tympanic membrane, middle ear or inner ear.

Daily intakes of 232Th and 238U in Japanese males.

Diet samples were collected by a duplicate portion method from 31 locations in Japan and were analyzed by inductively coupled plasma mass spectrometry. Average daily intakes per adult male were estimated at 1.7 mBq for 232Th and 8.8 mBq for 238U.

Sonic analysis for detection of cervical and intracranial vascular disease.

A sonic analysis system was developed for the detection of cervical and intracranial vascular disease. The system analyzes recorded sound signals converted to digital data, and plots the frequency, time interval after the QRS wave of the electrocardiogram, and amplitude on the graph using contour lines. Sonic analysis of 22 patients with and 23 patients without cerebrovascular disease identified a characteristic pattern of disease called the "circular pattern." Five of 10 patients with internal carotid artery stenosis, four of seven with cerebral aneurysms, three with cerebral arteriovenous malformation, and two of two with dural arteriovenous malformation showed the circular pattern. Only one of 23 control patients showed the circular pattern. This system is a promising method for cost-effective mass screening for the early detection of cerebrovascular disease.

[Evaluation of excised loach skin for studies on transdermal permeation of drugs in vitro].

Transdermal permeation of drug across the excised skin of Asian pond loach (Misgurnus anguillicaudatus Cantor) was studied in vitro. Ten kinds of drugs, D-glucose, L-glucose, sucrose, salicylic acid, cefazolin, urea, antipyrine, naphazoline, propranolol and enviomycin were permeated through the excised loach skin, whereas dextran, a polysaccharide, could not be permeated through the skin. The observed transdermal flux (J), the slope of the line obtained from plots of the cumulative amount of drug permeated vs. time, increased as the drug concentration in the donor compartment solution (50----300 mM), as the temperature of the skin (test solution) increased (4----37 degrees C), or as the molecular weight of compound decreased. These results suggested that the transport of drug across the loach skin was due to the passive diffusion mechanism. The effect of pH on the permeation of drug could not be elucidated since the loach skin was drastically impaired below pH 5.0. The J values of antipyrine and naphazoline varied in proportion to the drug concentration in the donor compartment solution at time zero (C0). The permeability constants (Kp, cm.h-1) of antipyrine and naphazoline were 12.9 x 10(-2) and 9.2 x 10(-2), respectively. However, the J values of salicylic acid and urea were not proportional to C0 in high concentration (300 mM). Probably, these results were related to the irritative effect of salicylic acid or urea on the skin.

[A complete response persisting for twelve months with the use of TS-1 in a patient with paraaortic lymph node metastasis of gastric cancer].

A 74-year-old female patient underwent total gastrectomy, splenectomy and D2 lymph node dissection for gastric cancer with non-dissectible paraaortic lymph node metastasis. Pathological examination revealed a high level of metastasis of dissected lymph nodes. The patient received daily oral administration of 100 mg TS-1, a novel oral anticancer agent. Each treatment course consisted of a four-week administration followed by two drug-free weeks. A partial response was obtained after the second course and a complete response was observed in the middle of the fourth and after the sixth course. The treatment was stopped because of grade 2 anemia in the middle of the seventh course, but no other adverse effect was observed. Complete response of the treatment persisted for twelve months and the patient has now been in good health without a recurrence for twenty months after surgery. Although the prognosis of gastric cancer with a high level of lymph node metastasis is poor, TS-1 therapy may have a potent efficacy in gastric cancer patients with a high level of lymph node metastasis such as the current case.