Expression pattern of REIC/Dkk-3 in mouse squamous epithelia.

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.

Role of intraoperative enteroscopy for surgical decision making with Crohn's disease.

BACKGROUND: This study aimed to assess the role of intraoperative enteroscopy (IOE) in determining surgical treatment. METHODS: The IOE procedure was performed for 30 patients with Crohn's disease. The degree of stricture and the presence of active ulcer were examined. Preoperative diagnoses and intraoperative findings obtained by inspection and palpation were noted and compared with the IOE findings. RESULTS: Of the 78 intestinal strictures observed by IOE (42%), 33 were not found by preoperative examination. Of the 45 strictures confirmed by IOE to be severe (<15 mm in diameter), 8 were judged to be mild (15-25 mm in diameter) or were not even identified by intraoperative inspection and palpation. Active ulcer was found at 12 of 33 mild strictures, and all 12 strictures were surgically corrected. Of 11 severe strictures detected by IOE at previous surgical sites, 9 were found preoperatively, and 4 were judged to be mild on the basis of inspection and palpation. Stricture was found at the ileocecal valve by IOE in seven patients, but was not diagnosed preoperatively in two of these patients. CONCLUSION: Intraoperative enteroscopy provides useful information regarding the status of the lumen in patients with Crohn's disease.

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model.

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.

9-Hydroxyellipticine inhibits telomerase activity in human pancreatic cancer cells.

There is increasing interest in identifying potent inhibitors of telomerase because the enzyme plays a crucial role in the development of cellular immortality and carcinogenesis. We hypothesized that 9-hydroxyellipticine (9-HE), an antitumor alkaloid, would inhibit telomerase activity because the drug has a unique mechanism of inhibiting phosphorylation of mutant p53 protein via inhibition of protein kinases, thereby restoring wild-type p53 function. This study was conducted to examine the effect of 9-HE on telomerase activity in human pancreatic cancer cells with differing p53 gene status. 9-HE treatment at relatively high concentrations resulted in rapid, complete inhibition of telomerase activity, irrespective of the p53 status. We conclude that 9-HE may exert a strong inhibitory effect on telomerase activity possibly through inhibition of protein kinases rather than through restoration of functional wild-type p53.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation.

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential.

We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Genetic and molecular analyses revealed high telomerase activity and a mutation in the K-ras oncogene. Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors. Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood. Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death. These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.

Diverse effects of 9-hydroxyellipticine on the chemosensitivity of human pancreatic cancer cells harboring p53 mutations.

Recently, it has been shown that 9-hydroxyellipticine (9-HE), an antitumor alkaloid has a unique property of restoring functional wild-type (wt) p53 activity via inhibition of mutant (mt) p53 protein phosphorylation. In the present study, we investigated the effect of 9-HE on the drug sensitivity of human pancreatic cancer cells. Exposure of cells to 9-HE at a relatively low concentration of 1 microM induced almost no cell death but was sufficient to restore wt p53 activity, as evidenced by an induction of endogenous p21WAF1/CIP1 concomitant with G1 and G2/M arrests in cell-cycle progression. Pretreatment with 1 microM 9-HE markedly enhanced cell killing when combined with cisplatin or mitomycin C. In contrast, 9-HE pretreatment protected cells from killing by 5-fluorouracil, VP-16, or vincristine. These effects of 9-HE were specific for several cell lines containing mt p53 and were not observed in p53-negative or wt p53 expressing cells. Taken together, these findings suggest that 9-HE may exert different effects on the drug sensitivity of pancreatic cancer cells displaying p53 mutations possibly through restoration of wt p53.

Significance of cardiovascular malformations in cystic hygroma: a new interpretation of the pathogenesis.

Fetuses with cystic hygroma or loose skin of the neck were studied chromosomally and phenotypically to clarify the relation between neck abnormality and cardiovascular malformations. Of 12 fetuses, 9 had chromosome abnormalities: 4 with 45,X, 3 with trisomy 21, one each with trisomy 13, dup 6q. One had normal chromosomes. Two cases, in which chromosome analysis was unsuccessful, were morphologically suspected to be trisomy 13. Nine of the 12 fetuses had either bilateral cystic hygroma of the neck (7 cases) or nuchal bleb (2 cases: trisomy 13 and dup 6q). Two of the 3 remaining cases (trisomy 21) had loose skin of the neck, and one had edematous swelling of the skin of the neck. Except for the last case of trisomy 21, 11 fetuses (91.7%) had severe and/or rare cardiovascular malformations. They were divided into 3 major groups: a) spectrum of hypoplastic left heart syndrome (45,X and dup 6q), b) double outlet right ventricle, agenesis of semilunar valve (trisomy 13), and c) abnormality of atrioventricular orifice or valves (trisomy 21). One fetus with normal chromosomes had persistent left superior vena cava instead of absent right one and calcification of myocardium. Histological observation of edematous skin demonstrated the abnormal distribution of lymph vessels, including their absence. Some cases showed hypoplastic thymus. To integrate the findings of the present study and the descriptions in the literature, a pathogenesis is hypothesized in relation to migration of neural crest cells and extracellular matrix.

Detection of alpha-interferon in nasopharyngeal secretions and sera in children infected with respiratory syncytial virus.

We investigated the relationship between respiratory syncytial virus (RSV) antigen and interferon (IFN) in nasopharyngeal secretions (NPS) and sera obtained from 252 patients infected with RSV. A total of 146 (57.9%) of 252 patients had IFN in NPS with a mean titer of 28 units/ml and IFN was detected in 164 (71.6%) of 229 patients in the acute stage sera with a mean titer of 28 units/ml. IFN activities were neutralized with antiserum to IFN-alpha. RSV antigen in NPS decreased on Day 5 and later in parallel with the change of mean titer of IFN in NPS. IFN in NPS was detected in 40 to 60% of the samples with some fluctuation in the acute stage. Within 4 days IFN was detected in more than 70% of the sera whereas on Day 5 and later the IFN positivity rate decreased in sera. RSV antigen in NPS decreased in the older patient groups. No significant change of positive rate of IFN in NPS was observed in different age groups and the mean titer of IFN in NPS and sera did not vary with age, except in those younger than 3 months and older than 3 years of age.

Interferon production during the course of Mycoplasma pneumoniae infection.

In patients infected with Mycoplasma pneumoniae the development of interferon (IFN) was studied in nasopharyngeal secretions and sera. The production of IFN-gamma by lymphocytes was also investigated in response to M. pneumoniae antigen and mumps virus antigen. IFN-alpha was detected in 25 (61.0%) of 41 nasopharyngeal secretion samples and in 25 (59.5%) of 42 serum samples within 6 days after the onset of illness. IFN-alpha was significantly higher in nasopharyngeal secretions than in sera and a significant correlation was observed between the two. In most of the patients lymphocytes produced a larger amount of IFN-gamma in the convalescent stage than in the acute stage, when lymphocytes were stimulated with M. pneumoniae antigen. In some patients, however, lymphocytes did not produce IFN-gamma during the course of illness. Such lymphocytes, negative for IFN-gamma production in response to M. pneumoniae, produced IFN-gamma after the depletion of macrophages, and readdition of macrophages suppressed the production of IFN-gamma by lymphocytes. When lymphocytes were stimulated with heterogeneous antigen (mumps virus), they produced no IFN or a small amount of IFN in the acute stage of M. pneumoniae infection, and IFN production increased in the convalescent stage. Different mechanisms seem to work for homogeneous and heterogeneous antigens in the suppression of IFN production in M. pneumoniae infection.

Inhibition of the production of rat cytokine-induced neutrophil chemoattractant (CINC)-1, a member of the interleukin-8 family, by adenovirus-mediated overexpression of IkappaBalpha.

Cytokine-induced neutrophil chemoattractant (CINC)-1, a counterpart of the human growth-regulated gene product (GRO) of the interleukin-8 family, has been suggested to play critical roles as a mediator of inflammatory reactions with neutrophil infiltration in rats. NF-kappaB has been speculated to be involved in the production of CINC-1, since the NF-kappaB-binding domain is important for the CINC-1 promoter activity in several of our reporter assays. In the present study, we examined the effects of an overexpression of IkappaBalpha, a specific natural inhibitor of NF-kappaB, on CINC-1 production. For this purpose, we constructed two recombinant adenoviruses, AxCAIkappaBalpha and AxCAmutantIkappaBalpha, which express respectively wild IkBa and a mutated nondegradable IkappaBalpha in which serine residues 32 and 36 are replaced by alanine residues. Transfecting wild and mutant IkBa by these adenovirus vectors inhibited NF-kappaB activation and CINC-1 production, which were both caused by IL-1beta stimulation in the normal rat kidney epithelial cell line NRK-52E. We also showed that the nondegradable mutant IkappaBalpha was approximately 30 times more potent than the wild type in inhibiting CINC-1 production. These findings demonstrate that CINC-1 production with NF-kappaB activation is primarily regulated by non-phosphorylated IkBa in the cytoplasm.

Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells.

Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers.

Effect of lead on electrophoretic mobility of rat erythrocytes.

Lead often affects the erythrocyte membrane. The relationship between the changes in erythrocyte membrane and the anemia caused by lead is still unclear. Initially, the effect of lead injected intraperitoneally on the electrophoretic mobility of rat erythrocytes was investigated in order to study the relationship between them. As indices of lead exposure, hemoglobin (Hb) levels, hematocrits (Ht), delta-aminolevulinic acid dehydratase (ALA-D) activities and blood lead (blood Pb) levels in the injected rats were also examined. Exposure to lead significantly decreased the mobility of rat erythrocytes. The changes in mobility seemed to be less sensitive than those in ALA-D activity, however, the decreases in mobility were simultaneous with or prior to those in Hb level and Ht. The decreases in mobility were evident to some extent below a blood Pb level of 100 micrograms/100 ml and generally present at a level of 100 micrograms/100 ml and over. In the rats exposed to lead a significant negative correlation was found between the mobilities and the logarithms of blood Pb level.

Enhancement of drug-induced apoptosis by antisense oligodeoxynucleotides targeted against Mdm2 and p21WAF1/CIP1.

The p53 tumor suppressor gene plays an important role in DNA damage-induced apoptosis and, in general, inactivation of p53 contributes to poor response to chemotherapy. Apoptotic activity of p53 may be negatively modulated by expression of its downstream mediators, including Mdm2 and p21WAF1/CIP1. Consequently, these cellular pathways also represent potential targets for cancer therapy. This study investigated the effect of antisense oligodeoxynucleotides (ODNs), targeted against Mdm2 and p21WAF1/CIP1 on drug-mediated cell killing. Exposure of U2-OS osteosarcoma cells to DNA damaging agents, cisplatin or mitomycin C, caused upregulated expression of Mdm2 and p21WAF1/CIP1. Transient transfection of cells with antisense ODNs to Mdm2 mRNA inhibited Mdm2 protein expression and markedly enhanced apoptotic cell death induced by these drugs. Moreover, when p21WAF1/CIP1 expression was blocked by antisense transfection, drug-mediated cell killing was further accelerated. These results suggest that the enhanced expression of Mdm2 and p21WAF1/CIP1 may inhibit p53-mediated apoptosis and render cells resistant to the effects of DNA damaging agents. Consequently, antisense ODNs targeted against Mdm2 and p21WAF1/CIP1 could be employed in a potential therapeutic strategy sensitizing tumor cells to certain antineoplastic agents.

An experimental study of the effects of lead acetate on hearing. Cochlear microphonics and action potential of the guinea pig.

Guinea pigs were poisoned with repeated i.p. injections of 1% lead acetate. After 5 weeks, the animals were examined electrophysiologically by using cochlear microphonics (CM) and action potential (AP). The thresholds of maximum voltage of N1 in the AP of the animals injected with a total of 100 mg lead acetate were elevated about 15 dB and increased N1 latency was also observed. However, no significant changes in those of CM were found. The results suggest that lead acetate not only induces damage to the peripheral nerves, but also to the cranial nerves.

Effects of toluene exposure on blood pressure and its responsiveness to impulse noise in rats.

Effects of a single exposure to toluene at a concentration of 1000 for 4 h on rats' systolic blood pressure were investigated. 9 rats were used in each group (control and exposed groups). The basic blood pressure of the exposed group was significantly lower than that of the control group. The movement of systolic blood pressure due to impulse noise exposure was significantly smaller in the exposed group than in the control group. Toluene exposure at concentration of 1000 ppm for 4 h produced narcotic and inhibitory actions in the diencephalohypophysial and the adrenal gland system in rats.

Effects of toluene exposure on the rest-activity cycle of rats.

Rats were exposed to toluene at a concentration of 1000 ppm, 6 h/day, 6 times weekly, for 4 weeks. Functional disturbance of the circadian rhythm of spontaneous activity was found after repeated exposure to toluene, although single exposure under the same conditions did not influence the rhythm. The disturbance was characterized by a significant increase in distribution of activity in the "Light' (L) period, and a significant relationship was observed between the total number of days of exposure and the activity level in the L period. The toluene concentration in the blood immediately after the exposure was 6.8-7.2 microgram/ml after one and 18 days of exposure, respectively.

Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus.

Staphylococcus hyicus was isolated from healthy pigs and pigs affected with exudative epidermitis (EE). Thirty seven strains (P-7 to P-43) were isolated from pigs affected with EE on 8 farms while 131 strains were isolated from healthy pigs bred on 2 farms in Japan. Isolation rate for pigs affected with EE was 100% while that for healthy pigs was 35.4%. The biochemical and cultural characteristics of the isolates from healthy and diseased pigs were identical except for the Voges-Proskauer reaction. The culture supernatant of many isolates caused skin exfoliation in 1-day-old chickens. Therefore, many isolates were considered to produce S. hyicus exfoliative toxin (shET). The rate of shET production by the isolates from piglets affected with EE was 87.5%, while that of the isolates from healthy pigs was 76.1%. shETs were divided in two serotypes by immunodiffusion. Piglets experimentally infected with shET-producing and nonproducing strains were observed. Local skin erythema at the inoculation site was observed with nonproducing strains and disappeared within 48 h, while the skin erythema at the sites inoculated with shET-producing strains did not disappear until 7 days after inoculation. Typical clinical signs, such as exfoliation, exudation and crusting were observed only in the piglets inoculated with shET-producing strains.

Measurements of oxygen pressure in the vocal fold during laryngeal nerve stimulation.

To clarify the mechanisms of ischemic change in the vocal fold on phonation, model experiments were performed under various conditions by recording tissue oxygen pressure (Pto2) values under electrical stimulation of either the recurrent laryngeal nerves or external branches of the superior laryngeal nerves. In addition, autonomic nerve regulation of laryngeal vessels was estimated under administration of succinylcholine chloride. Oxygen pressures of the vocal fold were measured in the lamina propria and vocal muscle at the middle of the membranous portion. The Pto2 in the lamina propria showed a slight increase during lower-frequency stimulation and a slight decrease during higher-frequency stimulation of the recurrent laryngeal nerve. The Pto2 in the vocal muscle showed a decrease during stimulation of the recurrent laryngeal nerve. On the other hand, the Pto2 showed almost no change in the lamina propria or vocal muscle during stimulation of the external branch of the superior laryngeal nerve. It is supposed that an ischemic change in the vocal muscle on phonation results mainly from contraction of the vocal muscle. On the contrary, the ischemic change in the vocal mucosa on phonation does not appear to be directly caused by the contraction of the vocal muscle but by other factors, such as wavelike movement.

An experimental study of the circulation of the vocal fold on phonation.

The ischemic change of the vocal fold on phonation was studied with an oxygen electrode using the canine vocal fold. Oxygen pressures of a limited area of the vocal fold were measured in the lamina propria and vocal muscle at the middle of the membranous portion. The results are summarized as follows: oxygen pressure in each area decreased on phonation as compared with respiratory movement, an ischemic change of the vocal fold occurred on phonation, and the response in the lamina propria was less than that of the vocal muscle.