Effects of professional oral health care on elderly: randomized trial.

OBJECTIVE: To better understand the role of the professional oral health care for elderly in improving geriatric oral health, the effects of short-term professional oral health care (once per week for 1 month) on oral microbiological parameters were assessed. METHODS: Parallel, open-labelled, randomize-controlled trial was undertaken in a nursing home for elderly in Shizuoka, Japan. Thirty-four dentate elderly over 74 years were randomly assigned from ID number to the intervention (17/34) and control (17/34) groups. The outcomes were changes in oral microbiological parameters (number of bacteria in unstimulated saliva; whole bacteria, Streptococcus, Fusobacterium and Prevotella: opportunistic pathogens detection: and index of oral hygiene evaluation [Dental Plaque Index, DPI]) within the intervention period. Each parameter was evaluated at before and after intervention period. Four elderly were lost from mortality (1), bone fracture (1), refused to participate (1) and multi-antibiotics usage (1). Finally, 30 elderly were analysed (14/intervention and 16/control). RESULTS: At baseline, no difference was found between the control and intervention groups. After the intervention period, the percentage of Streptococcus species increased significantly in the intervention group (Intervention, 86% [12/14]; Control, 50% [8/16]: Fisher's, right-tailed, P < 0.05). Moreover, DPI significantly improved in the intervention group (Intervention, 57% [8/14]; Control, 13% [2/16]: Fisher's, two-tailed, P < 0.05). The improvement in DPI extended for 3 months after intervention. None of side effects were reported. CONCLUSION: The short-term professional oral health care can improve oral conditions in the elderly.

Development of in-vessel components of the microfission chamber for ITER.

Microfission chambers (MFCs) will measure the total neutron source strength in ITER. The MFCs will be installed behind blanket modules in the vacuum vessel (VV). Triaxial mineral insulated (MI) cables will carry signals from the MFCs. The joint connecting triaxial MI cables in the VV must be considered because the MFCs and the MI cables will be installed separately at different times. Vacuum tight triaxial connector of the MI cable has been designed and a prototype has been constructed. Performance tests indicate that the connector can be applied to the ITER environment. A small bending-radius test of the MI cable indicates no observed damage at a curvature radius of 100 mm.

Lipid peroxidation end products-responded induction of a preneoplastic marker enzyme glutathione S-transferase P-form (GST-P) in rat liver on admistration via the portal vein.

The in vivo induction mechanism of a preneoplastic marker enzyme, glutathione S-transferase P-form (GST-P), by a number of carcinogens and some noncarcinogens such as anti-oxidants [Proc. Natl. Acad. Sci. U.S.A. 85 (1984) 3964] has remained to be solved. Among the various administration routes tested, GST-P became immunohistochemically demonstrable in the liver centrilobular zone 3 after 24-48h on administration of prostaglandin J2's, 15-deoxy-Delta(12,14)-PGJ2, PGJ2 and Delta(12)-PGJ2 to male rats via the portal vein, whereby the animals had been pretreated with Soya oil intraperitoneally to exhaust fatty acid binding proteins. Unsaturated aldehydes, 4-hydroxynonenal, crotonaldehyde and acrolein, given by the same route induced putatively preneoplastic single cells positive for GST-P. As these lipid peroxidation end products are the substrates as well as inducers of the enzyme, its physiological function could be their detoxication. These results indicate that GST-P expression can be mediated through lipid peroxidation possibly accounting for induction observed with a wide variety of carcinogens. In addition, present method may also be of use as a direct, simple, rapid, and sensitive in vivo test in examination of other biological responses.

KAI1, CAR, and Smad4 expression in the progression of colorectal tumor.

PURPOSE: The abnormal expression of several genes is involved in the development of colorectal tumors. The aim of this study was to examine the expression of KAI1, CAR, and Smad4, which has been associated with the progression of other cancers. METHODS: We examined 12 adenomas, 38 primary carcinomas, 10 liver metastases, and 7 adenocarcinoma cell lines, by fluorescent multiplex reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: KAI1 expression was higher in adenomas (2.55 +/- 0.61; P < 0.05) and primary carcinomas (3.30 +/- 0.42; P < 0.005) than in normal mucosa, but it was not correlated with tumor stage. CAR expression was lower in Dukes' B (1.28 +/- 0.53) than in Dukes' A (2.38 +/- 0.48) (P < 0.05), but increased with tumor progression; Dukes' C (1.45 +/- 0.25), Dukes' D (1.53 +/- 0.14), and metastases (2.09 +/- 0.39) (P < 0.05). Smad4 expression increased in adenomas (2.30 +/- 0.46; P < 0.05), but decreased with tumor progression; Dukes' A (2.14 +/- 0.37), Dukes' B (1.65 +/- 0.41), Dukes' C (1.57 +/- 0.25), Dukes' D (1.08 +/- 0.18), and metastases (0.82 +/- 0.21) (P < 0.05). CONCLUSIONS: These results suggest that the upregulation of CAR and down-regulation of Smad4 are associated with the progression of colorectal tumors, while the upregulation of these genes and of KAI1 seems to be involved in the early stage.

Diagnostic application of CD44 variant expression in pancreatic juice for detection of pancreatic neoplasm.

Recently, increased and disorganized expression of CD44 variant exons (CD44v) has been demonstrated in several types of human malignancy. We tried to investigate CD44v expression in pancreatic juice from patients who underwent endoscopic retrograde pancreatography. We analyzed 24 patients with pancreatic neoplasms diagnosed histologically (adenocarcinoma, 17; adenoma, 7) and 15 patients with non-neoplastic lesions. The expression of CD44v mRNA in pancreatic juice was detected by using the reverse-transcription polymerase chain reaction technique followed by Southern hybridization with exon-specific probes. Of 17 patients with adenocarcinoma, 14 (82%) showed expression of CD44v6 mRNA and 11 (65%) showed expression of CD44v2 mRNA. Of 7 patients with adenoma, 6 (86%) were positive CD44v6 mRNA expression and 2 (29%) for CD44v2 mRNA expression; while, out of 15 patients with non-neoplastic lesion, 5 (33%) showed positive findings for CD44V6 mRNA and 3 (20%) for CD44v2 mRNA. Comparing of diagnostic accuracy among CD44v6, CD44v2 and cytological examination, the sensitivities for adenocarcinoma were 82%, 65% and 41% respectively. However, the specificity was lower in CD44v6 (50%), CD44v2 (77%) than in cytology (100%), because CD44v was positive in adenoma cases and normal cases. A combination of RT-PCR analysis for the expression of CD44v with cytological examination in the pancreatic juice may increase the accuracy of diagnosis for pancreatic cancer.

Alterations in expression of E2F-1 and E2F-responsive genes by RB, p53 and p21(Sdi1/WAF1/Cip1) expression.

RB, p53 and p21(Sdi1/WAF1/Cip1) interact in the induction of G1 arrest. We established osteosarcoma cell lines in which a tetracycline-regulatable promoter controls the induction of RB, p53 and p21. By using these cell lines, we investigated whether RB, p53 or p21 regulates, in the same manner or differently, expression and function of E2F-1 and its responsive genes. E2F-1 gene products and transcripts of the E2F-responsive genes decreased in response to RB. Similar changes occurred to p53 and p21 when RB is present. However, in the absence of RB, some of the E2F-responsive genes decreased in response to p53 but not to p21. Thus, RB is a critical component for regulating the E2F-responsive genes, while p53 alone affects only a subset of these genes.

Differences in carcinogenesis by the length of carcinogen exposure period in rat colon.

To clarify the carcinogenic factors--whether it is the kind of carcinogen or their length of exposure--that determine whether colorectal cancer develops from an adenoma or develops de novo in the absence of an adenoma, we histopathologically analyzed a total of 229 rat colon tumors induced by administration of 1,2-dimethyl-hydrazine (DMH) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for three or 15 weeks. In the three-week-exposure groups, 71% of DMH-induced carcinomas and 82% of MNNG-induced carcinomas coexisted with low-grade dysplasia (adenomatous remnant). However, in the 15-week-exposure groups, lowgrade dysplasia was observed in only 10% of DMH-induced and 27% of MNNG-induced carcinomas. Even in the tumors smaller than 20 mm3, it was observed in only 10% of DMH-induced and 32% of MNNG-induced carcinomas. Furthermore, carcinomas without low-grade dysplasia predominated from the initial period of tumor occurrence. Next, we investigated association of K-ras and APC gene mutations with these carcinogenesis patterns in 80 tumors. K-ras mutations were not detected in any tumors induced by three weeks of exposure. However, in the 15-week-exposure groups, this mutation was observed in 57% of DMH-induced tumors and 13% of MNNG-induced tumors. APC mutations in the region homologous to the human mutation cluster region were observed in only 6% of tumors. Thus, our results suggest that the carcinogenesis patterns in rat colon are dependent on the length of exposure to carcinogen and that K-ras mutations were partly involved in a subset of them.

Apoptosis-inducing activity of a driselase digest fraction of green tea residue.

We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70% ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.

Induction of apoptosis and cell cycle arrest in mouse colon 26 cells by benastatin A.

Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian glutathione transferases (GSTs). Since GST inhibitors such as ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of benastatin A on the survival of mouse colon 26 adenocarcinoma cells was compared with that of ethacrynic acid. When cells in stationary phase were treated with benastatin A, viable cells were found to be dose-dependently decreased after 3 days. In the case of ethacrynic acid, this became apparent within 24 h. Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases. The dominant GST in colon 26 cells was identified as the class Pi-form (GST-II), and the activities in crude extracts as well as purified GST-II were almost completely inhibited by 50 microM ethacrynic acid. Immunoblot and northern blot analyses revealed increased GST-II protein and mRNA levels in cells treated with ethacrynic acid. Benastatin A did not significantly affect the activity in the crude extract even at 20 microM, a 10-fold higher concentration than that which almost completely inhibited the activity of purified GST-II. However, GST activity and GST-II protein were decreased in colon 26 cells treated with benastatin A for 5 days, no significant activity being detected in the range of 16 - 20 microM. In addition, beta-actin and bax mRNAs were also decreased in a dose-dependent manner. Furthermore, flow cytometric analysis of colon 26 cells revealed that benastatin A blocked the cell cycle at the G1/G0 phase. Thus, benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.

Blood flow and leukocyte adhesiveness are reduced in the microcirculation of a peritoneal disseminated colon carcinoma.

Dynamic behavior of leukocytes in the microcirculation of solid tumor tissue was visualized using a fluorescent labeling technique combined with the use of a real-time confocal laser-scanning microscope (CLSM) system. Colon tumor cells (RCN-9) were inoculated into the peritoneal cavity of male Fischer 344 rats. Tumor-free rats were similarly injected with physiological saline (intraperitoneally). Ten days after tumor inoculation, the mesentery was exteriorized and subjected to vital microscopic observation under the CLSM system. Leukocytes were labeled with rhodamine 6G (100 microg kg(-1), intravenously), and their behavior within the microvessels (10-30 microm in diameter) was analyzed both in the solid tumor tissues and the normal mesentery. Wall shear rate was calculated from the measured values of vessel diameter and erythrocyte flow velocity. In tumor microvasculature of tumor-bearing rats, the centerline erythrocyte velocity (0.73 +/- 0.58 mm s(-1), mean +/- standard deviation) and wall shear rate (210 +/- 151 s(-1)) were significantly lower than those of the tumor-free rats (1.27 +/- 0.83 mm s(-1), 344 +/- 236 s(-1), respectively). Despite such reduced flow conditions, flux of the rolling leukocytes as well as density of the adhered leukocytes both decreased significantly in tumor microvasculature as compared with normal controls. The methods developed in this work show promise in improving our understanding of tumor biology and pathophysiology.

Identification of glutathione S-transferase p-1 as the class pi form dominantly expressed in mouse hepatic adenomas.

To clarify which of the two genes for pi class glutathione S-transferases (GSTs) (p-1 and p-2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription-polymerase chain reaction (RT-PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non-adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p-1 and p-2 mRNA levels separately, the products of RT-PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p-2) and then resolved by electrophoresis. The p-1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p-2 mRNA levels were increased in the lesions as compared with those in the surrounding non-adenoma tissues. Recombinant p-1 and p-2 proteins were expressed in Escherichia coli. Unlike p-1, the p-2 protein did not show any significant activity towards 1-chloro-2,4-dinitrobenzene and did not bind to S-hexylglutathione-Sepharose despite immunological cross-reactivity. The dominant pi class form in adenomas could also be identified as p-1 by its binding to S-hexylglutathione-Sepharose. Single radial immunodiffusion analyses confirmed that the p-1 protein levels were in line with the mRNA findings, i.e., 1.9+/-0.3 mg/g adenoma as compared to 6.5+/-1.2 mg/g non-adenoma tissue for males and 2.2+/-0.6 mg/g as compared to 0.7+/-0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p-1 level and the contribution of p-2 is minimal.

Hepatocyte culture utilizing porous polyvinyl formal resin maintains long-term stable albumin secretion activity.

To investigate the effects of culture conditions on the maintenance of metabolic functions of cultured hepatocytes, long-term hepatocyte culture lasting 20 days was performed under two different culture conditions, i.e. stationary cultures utilizing porous polymer (polyvinyl formal (PVF) resin) as a substratum and conventional monolayer dish cultures without PVF. Metabolic activities specific to hepatocytes were evaluated in terms of ammonia metabolism, urea synthesis, and albumin secretion. Concerning ammonia metabolic and urea synthetic activities, no significant differences in maintenance of these activities were found between the two culture conditions, and these activities rapidly decreased with the elapse of the culture period, especially during the early stage of the experiments. However, after day 10, these activities in the stationary cultures were maintained at a slightly more favorable level than in the monolayer cultures. On the other hand, compared with ammonia metabolism and urea synthesis, stable and well-maintained albumin secretion of hepatocytes (60% of the activity in day 1) was exhibited in the stationary culture experiments, despite that this particular activity under the monolayer culture condition gradually reduced to a very low level (5.7% of that on day 1) at the end of the culture. From the morphological observations, hepatocytes immobilized in the PVF resin revealed individual spherical shapes without forming multicellular aggregation, and it was suggested that this characteristic structure contributed to good albumin secretion of hepatocytes. In conclusion, the advantages of the hepatocyte culture technique utilizing PVF resin over the conventional dish culture in maintaining some representative metabolic function specific to hepatocytes were clarified.

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression.

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.

Kinetic evaluation of beta-neoendorphin hydrolysis by the somatic and testicular isozymes of human angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) has both somatic and testicular isozymes, the former possessing two catalytically active domains, amino-terminal and carboxyl-terminal, while the latter has only the carboxyl-terminal one. We compared hydrolysis processes of the nonapeptide beta-neoendorphin by the two isozymes of human ACE. Both isozymes hydrolyzed the peptide to Tyr1-Gly2-Gly3 by the sequential removal of carboxyl-terminal dipeptides in three consecutive steps. The rate constant values for the second step, conversion of beta-neoendorphin1-7 to Leu-enkephalin, by the somatic isozyme in the presence of 10 or 200 mM NaCl were 4-fold higher than those for the first step, conversion of beta-neoendorphin1-9 to beta-neoendorphin1-7. The k(cat) values of the somatic isozyme for beta-neoendorphin1-7 were 2-fold higher than those for beta-neoendorphin1-9, indicating that beta-neoendorphin1-7 is more rapidly hydrolyzed than beta-neoendorphin1-9. The rate constant value for the second step at 10 mM NaCl was 5-fold higher than that for the testicular isozyme. Similar extent of difference was also observed in k(cat) values for beta-neoendorphin1-7 between the two isozymes. These results suggest that the amino-terminal domain of the somatic isozyme mainly contributes to the conversion of beta-neoendorphin1-7 to Leu-enkephalin at a low NaCl concentration. Optimal chloride concentrations for the individual steps of beta-neoendorphin1-9 hydrolysis differed between the two isozymes.

Differentiation induced by RB expression and apoptosis induced by p53 expression in an osteosarcoma cell line.

Multiple genetic alterations, including concurrent inactivation of RB and p53, occur frequently in several human cancers. To investigate the biological significance of RB and p53 gene inactivations, a wild-type RB or p53 cDNA expression vector regulated by tetracycline was introduced by stable transfection into an osteosarcoma cell line Saos-2, in which both the RB and p53 genes were inactivated. Induction of introduced RB expression resulted in suppression of cell growth, increased percentage of cells at the G0/G1 phase, and enlargement of the cells. Furthermore, activity of alkaline phosphatase was increased and expression of fibronectin was decreased, suggesting the induction of cell differentiation by RB expression. Induction of p53 expression also resulted in significant suppression of cell growth with slight accumulation of cells at the G0/G1 and G2/M phases. The cells were detached from culture dishes and the dead cell fraction increased. Furthermore, condensation of chromatin and DNA fragmentation were observed, suggesting the induction of apoptosis by p53. These results suggest that RB and p53 play different roles in carcinogenesis of osteoblast; RB inactivation releases cells from G0/G1 arrest and suppresses cell differentiation while p53 inactivation assists the cells to proliferate by repressing both apoptosis and cell cycle arrest at G0/G1 and G2/M.

Evaluation of DNA polymerase beta gene mutation as a genetic marker for colorectal carcinoma.

DNA polymerase beta is known to be involved in repair of DNA damage. Frequent mutation of its gene in the segment encoding amino acids 149-297 has been reported in colorectal cancer. To investigate whether mutation in this region is available as a genetic marker for colorectal cancer, 11 primary tumors and 4 liver metastases from 11 patients were examined by fragment length analysis and single-strand conformation polymorphism (SSCP) analysis of reverse-transcription polymerase chain reaction (RT-PCR) products. Although allelic imbalance in the p53 and DCC genes were observed in ten out of eleven primary tumors and all liver metastases using a dinucleotide repeat polymorphism, mutation was not detected in the DNA polymerase beta mRNA. Neither was it detected in seven colon cancer cell lines. Present results suggest that mutation in this region is uncommon in colorectal cancers and is not a useful genetic marker for colorectal cancer.

Induction of apoptosis but not G1 arrest by expression of the wild-type p53 gene in small cell lung carcinoma.

Multiple genetic alterations, including inactivation of the p53 and RB genes and loss of heterozygosity on chromosome 3p, occur commonly in small cell lung carcinoma (SCLC). To assess the biological significance of p53 inactivation in the development of SCLC, tetracycline (Tc)-inducible p53 expression plasmids were introduced into a SCLC cell line, N417, in which the p53 gene as well as the RB gene was inactivated. In the absence (induced) of Tc, cells transfected with the wild-type p53 gene formed colonies in 29-58% of those with a mutant p53 gene. However, wild-type p53 genes were expressed in 0 of 43 transfectants, whereas mutant p53 genes were expressed in 75% (36/48) of the transfectants, suggesting that the growth of SCLC cells was suppressed by the expression of the wild-type p53 gene. Thus, wild-type p53-inducible clones were further established by transfection in the presence (repressed) of Tc. The in vitro growth was significantly suppressed by the induction of wild-type p53 expression, and apoptosis but not G1 arrest was observed within 24 h of p53 induction. These results strongly suggest that the restoration of the p53 function is sufficient to suppress the growth of SCLC cells in which other genetic alterations remain uncorrected, and that growth suppression by p53 is due to induction of apoptosis but not due to induction of G1 arrest through the RB pathway.

Flow visualization of microcirculation in solid tumor tissues: intravital microscopic observation of blood circulation by use of a confocal laser scanning microscope.

To visualize the blood flow in the neovasculature of a solid tumor tissue, we utilized an intravital microscope system equipped with real-time confocal laser scanning optics. Fluorescent labeling of the blood cells and blood plasma enabled quantitative measurements of microhemodynamic parameters such as flow velocity, blood cell flux and vessel diameter in both the normal and tumor microvasculature.

Alternative splicing of the neurofibromatosis 1 gene correlates with growth patterns and neuroendocrine properties of human small-cell lung-carcinoma cells.

Two distinct transcripts, type I and type II, of the neurofibromatosis I (NFI) gene are generated by alternative splicing in the region corresponding to the gene's GTPase-activating protein-related domain (GRD). Relative expression levels of these 2 transcripts were previously correlated to neural differentiation. Since small-cell lung carcinoma (SCLC) often exhibits neuroendocrine properties, we analyzed the type-I to type-II mRNA ratio in 15 SCLC cell lines, using reverse transcriptase and polymerase chain reaction methods. The type-I mRNA was predominant in 10 cell lines; 8 of them grew as floating aggregates in culture and had high L-dopa decarboxylase (DDC) activity. The other 5 lines predominantly expressed type-II mRNA, adhered to the culture substrate, and expressed low or undetectable levels of neural cell-adhesion molecule (NCAM) antigen and DDC activity. N2+, one of the subclones of NCI-N417 cells, exhibited a higher type-I to type-II ratio after the cells had adhered to a laminin-coated plate and had emitted neurite-like processes. These findings provide evidence that alternative splicing patterns of NFI mRNA correlate with the mechanisms that regulate the growth patterns and neuroendocrine properties of SCLC cells in vitro.

Allelic losses associated with the metastatic potential of colorectal-carcinoma.

The aim of this study was to define the association of allelic losses with the metastatic potential of colorectal carcinoma and to determine whether allelic losses can be genetic markers for the prognosis of patients with colorectal carcinoma. Eighty primary colorectal tumors and 31 liver metastases from 95 patients were examined for loss of heterozygosity (LOH) at the APC, p53, RB, DCC and chromosome 14q loci by using polymerase chain reaction-single strand conformation polymorphism analysis and restriction fragment length polymorphism analysis. The incidence of LOH at the DCC and RB loci and on chromosome 14q in liver metastases was significantly higher than that in primary tumors. DCC and RB alterations were detected more frequently in primary tumors with higher metastatic potential. Although no statistically significant association was found between these losses and survival or distant metastasis, patients with DCC losses showed poorer survival by multivariate analysis (p=0.056). Thus, inactivation of the DCC and RB genes and gene(s) on chromosome 14q seem to be critical genetic events for the acquisition of metastatic potential in colorectal carcinoma. However, further studies will be required to utilize these genetic alterations as valuable prognostic markers.