Effects of Sulfonylureas on Periodontopathic Bacteria-Induced Inflammation.

Interleukin-1ß (IL-1ß) is an inflammatory cytokine produced by monocytes/macrophages and is closely associated with periodontal diseases. The NLRP3 inflammasome is involved in IL-1ß activation through pro-IL-1ß processing and pyroptotic cell death in bacterial infection. Recently, glyburide, a hypoglycemic sulfonylurea, has been reported to reduce IL-1ß activation by suppressing activation of the NLRP3 inflammasome. Therefore, we evaluated the possibility of targeting the NLRP3 inflammasome pathway by glyburide to suppress periodontal pathogen-induced inflammation. THP-1 cells (a human monocyte cell line) were differentiated to macrophage-like cells by treatment with phorbol 12-myristate 13-acetate and stimulated by periodontopathic bacteria, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum, in the presence of glyburide. IL-1ß and caspase-1 expression in the cells and culture supernatants were analyzed by Western blotting and enzyme-linked immunosorbent assay, and cell death was analyzed by lactate dehydrogenase assay. Stimulation of THP-1 macrophage-like cells with every periodontopathic bacteria induced IL-1ß secretion without cell death, which was suppressed by the NLRP3 inhibitor, MCC950, and caspase-1 inhibitor, z-YVAD-FMK. Glyburide treatment suppressed IL-1ß expression in culture supernatants and enhanced intracellular IL-1ß expression, suggesting that glyburide may have inhibited IL-1ß secretion. Subsequently, a periodontitis rat model was generated by injecting periodontal bacteria into the gingiva, which was analyzed histologically. Oral administration of glyburide significantly suppressed the infiltration of inflammatory cells and the number of osteoclasts in the alveolar bone compared with the control. In addition to glyburide, glimepiride was shown to suppress the release of IL-1ß from THP-1 macrophage-like cells, whereas other sulfonylureas (tolbutamide and gliclazide) or other hypoglycemic drugs belonging to the biguanide family, such as metformin, failed to suppress IL-1ß release. Our results suggest that pharmacological targeting of the NLRP3 pathway may be a strategy for suppressing periodontal diseases.

Anxiety and pain suppress the natural killer cell activity in oral surgery outpatients.

OBJECTIVE: Psychological stress has an influence on natural killer cell (NK) activity, which plays a central role in protection against microbial infection and cancer. Anxiety concerning cancer is a typical type of psychological stress observed in patients and is associated with various diseases. In this study, we examined whether anxiety about cancer reduces the NK activity or quality of life (QOL), or both, of outpatients. PATIENTS AND METHODS: NK activity was determined by means of a 4-hour 51 chromium-release assay, and a QOL questionnaire by the World Health Organization called QOL-BRIEF was also used. One hundred forty-four patients were asked a variety of questions about anxiety with respect to cancer and pain on their first visit to the outpatient clinic of our Department of Oral Surgery. RESULTS: The NK activity in patients with anxiety about cancer was significantly lower (P < .001) than that in those with no such anxiety. In contrast, NK activity was not influenced by any other factor tested, including differences in diseases and QOL scores. CONCLUSION: Anxiety about cancer may be one of the primary factors for suppression of NK activity in oral surgery outpatients.

[Colorectal cancer in spouses of colorectal cancer patients].

Married couples share home environments and life style for years. In the case of colorectal cancer, an association with insulin resistance was reported. We determined the presence of the insulin-resistance syndrome (IRS, 1 or more of the following: body mass index of > 25 kg/m2, diabetes, or hyperlipidemia) in 84 colorectal cancer patients, of whom 61 patients (73%) had IRS. The incidence of the distal colorectal cancer, which has been declining in the United States, was significantly higher in the IRS group than in the non-IRS group (75.4 vs 52.2%, p = 0.0400). Some mechanisms may promote the progression of mucosal lesions to invasive cancers in the distal colorectum. There were no significant differences with respect to the age (64.6 +/- 9.4 vs 64.3 +/- 11.3 yr, p = 0.8298), height (159 +/- 9 vs 157 +/- 8 cm, p = 0.1375), and body mass index (22.2 +/- 3.6 vs 22.4 +/- 2.7 kg/m2, p = 0.6364) between the patients and their spouses. In 84 couples in whom colorectal cancer develops at least in one may then not illustrate the nursery rhyme: "Jack Sprat could eat no fat, His wife could eat no lean...". The spouses had been married for an average of 38 years, and in 30 spouses who had been followed in a colorectal cancer screening, 5 developed colorectal cancer. To diminish the incidence of colorectal cancer in Japan, we might advise screening colonoscopy to the spouses of colorectal cancer patients, or déjà vu all over again?

Comparison of the inpatients admitted during the recent 5 years (January 1994-December 1998) with those admitted during the previous 5 years (January 1989-December 1993): a clinico-statistical study.

As society ages, the composition of the diseases that occur within it changes accordingly. With that in mind, we examined the characteristics and trends in the recent inpatients and compared these recent inpatients with those of a previous report to identify the changes that accompany the aging of society. Subjects were 1,534 cases (men 56.9%, female 43.1%, average age 47.1 years) who were hospitalized at Kurume University Hospital for treatment during the 5-year period from January 1st, 1994 through December 31st, 1998. The ratio of inpatients over 65 years old was about 1.8 times higher than in the previous study, showing a clear trend toward an increased overall age of inpatients. As for the types of disease observed, the most common malignancies were epithelial tumor, followed by other benign tumors, as well as 76 cases which included diseases resembling tumor (epulis and exostosis etc.). A majority of the patients (55.6%) were directed to the Hospital by their dentist, a finding similar to that of the previous report. As for geographical distribution, 93.3% of the inpatients lived within 40 km of the center of Kurume City where our oral surgery is located, an increase of about 10% from the last report. In other words, our results showed a reduction in the sphere of treatment distribution.

Characterization of a novel member of the FGF family, XFGF-20, in Xenopus laevis.

The cDNA for a novel member of the FGF family (XFGF-20) was isolated from a Xenopus cDNA library prepared at the tailbud stage using as a probe the product of degenerate PCR performed with primers based on mammalian FGF-9s. This cDNA was 1860 bp long, and contained a single open reading frame that encoded 208 amino acid residues. The deduced amino acid sequence contained a motif characteristic of the FGF family and it was similar (73.1% overall homology) to XFGF-9 but differed from XFGF-9 in its amino-terminal region (33.3% homology). XFGF-20 mRNA was expressed only zygotically in embryos at and after the blastula stage, but it was also specifically expressed in the stomach and testis of adults. By contrast, XFGF-9 mRNA was expressed maternally in eggs and in many adult tissues. When XFGF-20 mRNA was overexpressed in early embryos, gastrulation was abnormal and development of anterior structures was suppressed. In such embryos, the expression of the Xbra transcript was suppressed during gastrulation while the expression of the transcripts of cerberus, Siamois, dkk-1, chordin, and Xotx-2 genes was normal. These results suggest that correct expression of XFGF-20 during gastrulation is required for the formation of normal head structures in Xenopus laevis during embryogenesis and that expression of the Xbra gene mediates this phenomenon.

Human genes for KNSL4 and MAZ are located close to one another on chromosome 16p11.2.

KNSL4 (Kid; kinesin-like DNA-binding protein) is a member of the kinesin family that is involved in spindle formation and the movements of chromosomes during mitosis and meiosis. Myc-associated zinc finger protein (MAZ) participates in both the initiation and the termination of transcription of target genes. We isolated genomic DNA clones that encoded KNSL4 and MAZ from a human cosmid library. Sequence analysis revealed that the two genes were very close to one another. The distance between the two genes was only 1. 2 kb, and this intervening 1.2-kb region was extremely GC-rich. The gene for KNSL4 spanned 16 kb and consisted of 14 exons and 13 introns, while the gene for MAZ spanned 6 kb and consisted of 5 exons and 4 introns. The two genes were mapped to chromosome 16p11.2 by fluorescence in situ hybridization.

Requirement of expression of P-glycoprotein on human natural killer leukemia cells for cell-mediated cytotoxicity.

The requirement of P-glycoprotein, a product of the multidrug resistance (MDR)1 gene, for natural killer (NK) cell-mediated cytotoxicity was examined by using a human NK-like cell line, YTN, which is cytotoxic toward JY cells. YTN cells express P-glycoprotein, a judged by flow cytometry and polymerase chain reaction of reverse-transcribed mRNA. YTN cell-mediated cytotoxicity was inhibited by MDR-reversing reagents as well as the F(ab')2 fragment of a monoclonal antibody against P-glycoprotein. Furthermore, antisense oligonucleotides for MDR1 mRNA inhibited expression of P-glycoprotein as well as YTN cell-mediated cytotoxicity. Thus, this study provides firm evidence that P-glycoprotein plays an essential role in cell-mediated cytotoxicity.

Assignment of the human gene for KBF2/RBP-Jk to chromosome 9p12-13 and 9q13 by fluorescence in situ hybridization.

The transcription factor KBF2 has been characterized as a factor that binds to the NFkB site of mouse major histocompatibility complex (MHC) class I genes and its amino acid sequence has been shwn to be identical to those of members of the recombination signal-sequence binding protein (RBP-Jk) family. Previous studies by Amakawa et al. (Genomics 17, 306-315, 1993) demonstrated that the functional gene is localized at human chromosome 3q25. However, in the present study we showed by in situ hybridization with the functional KBF2/RBPJk cosmid clone that the gene is localized at 9p12-13 and 9q13, namely, at the same loci as pseudogenes that were reported previously (Zhang et al, Jpn J Human Genet 39, 391-401, 1994).

Cloning of Xenopus Dr1 (TBP-binding repressor) and its expression in oocytes and early embryos.

With a final goal to study how TBP-binding repressor Dr1 regulates transcription in Xenopus early embryos, we cloned its cDNA from Xenopus liver cDNA library. The cDNA was 1,986 bp long, with the open reading frame coding for 175 amino acids, whose sequence was highly homologous to its human counterpart. Xenopus Dr1 mRNA was expressed from the earliest stage of oogenesis, inherited as maternal mRNA at a high level, but its level became low at and after the neurula stage where RNA synthetic activity is strongly activated. Dr1 mRNA occurred in larger amounts in the animal half than in the vegetal half in 8-cell stage embryos, and in neurula and tailbud stage embryos its distribution was slightly larger in the anterior part than in the posterior part. These data show that Dr1 mRNA is expressed in a temporally and spatially regulated manner, and its occurrence at higher levels in earlier stage embryos may be responsible for their low transcriptional activity.

Cloning and expression studies of cDNA for a novel Xenopus cadherin (XmN-cadherin), expressed maternally and later neural-specifically in embryogenesis.

From a Xenopus tailbud cDNA library, we obtained the cDNA for a novel cadherin which was named as XmN-cadherin (Xenopus maternally expressed neural cadherin). The cDNA consisted of 3690 bp and encoded 922 amino acid residues. XmN-cadherin preserved five extracellular cadherin motifs, a single transmembrane domain, and a cytoplasmic domain, and was closely related by its sequence to R- and N-cadherin. In the adult frog, XmN-cadherin mRNA was detected strongly in ovary, testis, brain, eye, and kidney, and weakly in stomach, and intestine. In the egg, the mRNA occurred as a maternal mRNA at a relatively high level, and its level became very low by the neurula stage, then increased steadily thereafter. Dissection experiments with 8-cell stage and neurula stage embryos revealed that the maternally inherited mRNA was relatively uniformly distributed within the embryo. By a sharp contrast, whole mount in situ hybridization revealed that the zygotically expressed mRNA occurred almost exclusively in neural tissues such as brain, the anterior part of spinal cord, and the optic and otic vesicles. Thus, XmN-cadherin appears to have at least triple functions; it probably contributes in early embryos to cell-type non-specific cell adhesion, but in post-neurula embryos may be responsible for the development and/or maintenance of anterior neural tissues, and may be used in adult frog for the development and/or maintenance of neural, endodermal and reproductive organs.

Differential display analysis of gene expression in developing embryos of Xenopus laevis.

Differential display (DD), an arbitrarily primed RT-PCR fingerprinting technique, is a novel approach for the search of differentially expressed transcripts. Using our improved DD protocol, reproducible cDNA fingerprints were successfully obtained from RNAs of Xenopus laevis embryos at six representative stages. Parallel comparison among the fingerprints revealed a number of bands with differential expression patterns. Analysis with clones of three randomly chosen bands confirmed that their expression patterns were faithfully reflected on fingerprints, thereby proving the reliability and validity of the approach. Nucleotide sequencing of these clones revealed that one is identical with a known transcript (cardiac actin), the second is a novel developmentally regulated gene showing no significant homology with those reported previously, and the last is a close but unique relative of XK endo B gene showing somewhat different spatial expression pattern. These results indicated that the DD analysis provides a rapid and reliable way for the identification of novel differentially expressed genes as well as a unique 'scope' for the survey of the changes in overall gene expression profiles occurring in the early embryonic development of Xenopus as well as of other organisms.

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis.

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.

Effect of opioid peptides on dopamine release from nucleus accumbens after repeated treatment with methamphetamine.

The effect of opioid peptides on extracellular dopamine levels in the nucleus accumbens was compared between rats treated with methamphetamine and saline repeatedly (for 9 days) by using microdialysis. After the period of repeated treatment, the rats in both groups were kept for an additional 9 days without further treatment. Repeated administration of methamphetamine reduced the decreasing effect of dynorphin (10 microM), applied locally in the perfusate, and enhanced the increasing effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO, 10 microM) on the extracellular dopamine levels in the nucleus accumbens. It is possible that repeated treatment with methamphetamine leads to attenuation of the inhibition and enhancement of the stimulation of dopamine release from the nucleus accumbens via presynaptic dynorphin- and enkephalin-sensitive receptors, respectively.

A CRF antagonist attenuates stress-induced increases in NA turnover in extended brain regions in rats.

We investigated the effects of intracerebroventricular (i.c.v.) administration of corticotropin-releasing factor (CRF) antagonist, alpha-helical CRF9-41 (ahCRF), on increases in noradrenaline (NA) turnover caused by immobilization stress in rat brain regions. Pretreatment with ahCRF (50 or 100 micrograms) significantly attenuated increases in levels of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4), the major metabolite of NA in rat brain, in the locus coeruleus (LC) region, and attenuated the MHPG-SO4/NA ratio after immobilization stress for 50 min in the cerebral cortex, hippocampus, amygdala, midbrain and hypothalamus. However, stress-induced increases in plasma corticosterone levels were not decreased significantly by pretreatment with ahCRF. These results suggest that CRF, released during stress, causes increases in NA release in extended brain regions of stressed rats.

Muscimol-induced increase in dopamine release and metabolism is not observed in kainic acid-lesioned striatum of conscious rats: an in vivo microdialysis study.

Local application of muscimol through the striatal dialysis membrane caused a significant increase in both dopamine release and dopamine metabolism in the striatum of conscious rats, however, both elevations induced by muscimol were significantly lower in the kainic acid-lesioned striatum when assessed with in vivo brain microdialysis. These findings show that intra-striatal muscimol indirectly stimulates nigrostriatal dopaminergic function by possibly causing an inhibition of striatal gamma-aminobutyric acid neurons.