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Inflammatory responses triggered by activation of the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway are a key mechanism in nonsteroidal anti-inflammatory drug-induced enteropathy. The aim of this study was to investigate the probiotic effect of Lactobacillus casei strain Shirota (LcS) on indomethacin-induced small intestinal injury. Rats pretreated with viable LcS or heat-killed LcS once or once daily for a week were administered indomethacin by gavage to induce injury. Anti-inflammatory effects of L-lactic acid (1-15 mM) were evaluated in vitro by use of THP-1 cells. One-week treatment with viable LcS prevented indomethacin-induced intestinal injury with increase in the concentration of lactic acid in small intestinal content and inhibited increases in myeloperoxidase activity and expression of mRNA for tumor necrosis factor-alpha (TNF-alpha) while affecting neither TLR4 expression nor the number of gram-negative bacteria in intestinal content, whereas neither heat-killed LcS nor a single dose of viable LcS inhibited intestinal injury. Prevention of this injury was also observed in rats given l-lactic acid in drinking water. Both L-lactic acid and LcS culture supernatant containing 10 mM lactic acid inhibited NF-kappaB activation and increases in TNF-alpha mRNA expression and TNF-alpha protein secretion in THP-1 cells treated with LPS. Western blot analyses showed that both L-lactic acid and LcS culture supernatants suppressed phosphorylation and degradation of I-kappaB-alpha induced by LPS without affecting expression of TLR4. These findings suggest that LcS exhibits a prophylactic effect on indomethacin-induced enteropathy by suppressing the LPS/TLR4 signaling pathway and that this probiotic effect of LcS may be mediated by L-lactic acid.

We studied the relationship between the mitochondrial density in the cells and the cellular sensitivity to the toxicity of cis-diaminedichloroplatinum II (cisplatin), a potent anticancer agent. Biochemical analyses revealed that the density of mitochondria in the intestinal epithelium changed markedly along its entire length. The density was the highest at the duodenum, medium at the jejunum, and the lowest at the ileum. The sensitivity of epithelial cells to cisplatin toxicity was the highest at the duodenum, medium at the jejunum, and the lowest at the ileum as judged from the occurrence of apoptosis. Similar correlation between the cisplatin sensitivity and mitochondrial density was also observed with in vitro experiments, in which intestinal epithelial cells (IEC-6) and their rho0 cells with reduced number of mitochondria were used. The rho0 cells had a strong resistance to cisplatin compared with the control cells. Cisplatin markedly increased mitochondrial generation of reactive oxygen species in IEC-6 but not in rho0 cells. We analyzed the sensitivity of eight cell lines with different density of mitochondria to cisplatin and found the same positive correlation. These observations clearly show that cellular density of mitochondria is the key factor for the determination of the anticancer activity and side effects of cisplatin.

Gravity resistance is a response that enables plants to develop against the gravitational force. Hypergravity conditions produced by centrifugation have been used to analyze the mechanisms of gravity resistance responses. Under hypergravity conditions, plants construct short and thick shoots and increase cell wall rigidity for resisting the gravitational force. Hypergravity caused a decrease in the percentage of cells with transverse microtubules, and an increase in that with longitudinal microtubules. Such a prompt reorientation of cortical microtubules is involved in the changes in morphology of shoots by gravity. Hypergravity increased cell wall rigidity by increasing the molecular mass of xyloglucans via suppression of xyloglucan breakdown as well as by the thickening of cell walls. Blocker reagents of mechanoreceptors nullified the above-mentioned changes induced by hypergravity. Gravity resistance responses were brought about normally in mutants deprived of gravitropism. This result indicates that the graviperception mechanism in gravity resistance is independent of that in gravitropism. Gravity resistance responses were brought about independently of the direction of gravistimuli, but the responses disappeared in the presence of blockers of mechanoreceptors. Thus, in gravity responses, plants may perceive the gravitational force independently of the direction of stimuli by mechanoreceptors on the plasma membrane, and may utilize the signal to construct a tough body.

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.

Cell wall structure of wheat coleoptiles grown under continuous hypergravity (300 g) conditions was investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The amounts of cell wall polysaccharides substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. As a results, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. The major sugar components of the hemicellulose fraction, a polymer fraction extracted from cell walls with strong alkali, were arabinose (Ara), xylose (Xyl) and glucose (Glc). The molar ratios of Ara and Xyl to Glc in hypergravity-treated coleoptiles were higher than those in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. These results suggest that hypergravity stimuli bias the synthesis of hemicellulosic polysaccharides and increase the proportion of acidic polymers, such as arabinoxylans, in cell walls of wheat coleoptiles. These structural changes in cell walls may contribute to plant resistance to hypergravity stimuli.

We reported that elongation growth of plant shoots and roots is suppressed by hypergravity, with the rate decreasing in proportion to logarithm of the magnitude of gravity. In hypergravity-induced growth inhibition of shoots, graviperception is supposed to be independent of that in gravitropism and to involve mechanoreceptors. However, the graviperception mechanism in the hypergravity-induced growth inhibition of roots is not known. In the present study, we compared the mechanism in the hypergravity-induced growth inhibition of roots with that in gravitropism. The removal of root cap did not influence hypergravity-induced growth inhibition of roots, although the gravitropic curvature was completely inhibited. Hypergravity had no effects on growth of azuki bean roots in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. On the contrary, lanthanum or gadolinium at the same concentration did not influence gravitropism of roots. These results suggest that the graviperception mechanism in the hypergravity-induced growth inhibition of roots is independent of that in gravitropism. Hypergravity-induced growth inhibition of azuki bean roots was observed irrespective of the direction of stimuli, which disappeared in the presence of lanthanum or gadolinium. Thus, in the hypergravity-induced growth inhibition, roots may perceive the gravity signal by mechanoreceptors on the plasma membrane independently of the direction of stimuli, and may utilize it to regulate their growth rate.

Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.

The correlation of white light-induced changes in osmotic concentration in the coleoptile and the first leaf and the growth rate of these organs in maize seedlings, was examined in relation to sugar distribution and invertase activity. One hour irradiation with white light decreased the osmotic concentration in basal zones of the coleoptile and increased it in the apical zones of the first leaf. The change in the osmotic concentration was positively correlated with the growth rate of both organs. The amount of total osmotic solutes in each zone was highly correlated with that of soluble sugars. Light decreased the activity of wall-bound invertase in the coleoptile, but increased it in the first leaf. A high correlation existed between the content of soluble sugars and invertase activity in both organs. During 1 h incubation in the light, ca 2 µmol of soluble sugars per seedling was lost from the coleoptile and gained in the first leaf. Light promoted sugar exudation from the excised coleoptile, but the amount of soluble sugar exuded represented 5% of sugar loss from the coleoptile in intact seedlings. These results indicate that in maize seedlings white light controls the growth rate of the coleoptile and the first leaf through the osmotic concentration. Light may have an osmoregulatory function in the control of sugar distribution between the coleoptile and the first leaf by regulating the activity of wall-bound invertase.

We investigated the effects of serum-free conditioned medium (SFCM) of colon cancer cells on human umbilical vein endothelial cells (HUVECs). SFCM of highly liver metastatic cell line (LM-HS) strongly enhanced proliferation, migration and tube formation of HUVECs. Moreover, it was suggested that a main angiogenic factor derived from LM-H5 may be VEGF based on HPLC, SDS-PAGE analysis and neutralizing experiment using anti-growth factor antibodies. These findings suggest that angiogenesis is related to the processes of metastasis of colon cancer, and that VEGF synthesized by colon cancer cells acts as a key factor inducing angiogenesis.

Several cytokines, including IL-1, TNF, LIF and IL-6 have recently been proposed as cachexia inducers. We experimentally examined the participation of cytokines, particularly, IL-6, in cancer cachexia using the human digestive cancer cell lines MKN 28, MKN 45, MKN 74, Kato-III, OCUM-2M (gastric cancer), SW1990, Panc-1 (pancreatic cancer), and OCUG (gallbladder cancer). A high level of IL-6 was detected in the OCUG culture medium. Nude mice bearing OCUG tumor had reduced body weight even when the tumor was relatively small. Loss of both muscle and adipose tissue, anemia, hypoglycemia, and a high serum level of human IL-6 were observed in these mice. However, body weight recovered rapidly to the level of that of nontumor-bearing mice after resection of OCUG tumor. Antihuman IL-6 but not anti-murine IL-6 receptor antibodies significantly suppressed the development of cachexia as measured by various indicators of cachexia including loss of both muscle and adipose tissue, anemia and hypoglycemia, as well as weight loss. These results suggest that OCUG-bearing mice exhibited cancer cachexia mediated by IL-6, and that of OCUG cell line might be useful as a human digestive cancer cachexia model.

We investigated the inhibitory effects of intrasplenic combination therapy with OK-432 and recombinant interleukin-2 (IL-2) on liver metastasis of colorectal carcinoma. Intrasplenic administration group significantly inhibited the development of liver metastasis compared with subcutaneous administration group (p<0.05). Combination therapy significantly inhibited the development of subclinical liver metastasis compared with that in the control group. Combination therapy decreased the percentage of cells expressing CD8a, which may be a part of effective factors of combination therapy, and improved overall survival rate. These findings suggested intrasplenic combination therapy with OK-432 and IL-2 might be effective in inhibiting liver metastasis of colorectal carcinoma.

A new human cell line (OCUG-1) was established from peritoneal effusion of a patient with malignant gallbladder carcinoma. OCUG-1 cells proliferated mainly in clusters of cells partially floating in a monolayered sheet. The population doubling time is 47.1 h. DNA analysis showed that OCUG-1 cells were aneuploid and had two G(0)/G(1) peaks. The number of chromosomes was distributed in a broad range from 52 to 139. Subcutaneous injections of the cells induced tumor formation in all nude mice. The reconstituted tumors were poorly differentiated adenocarcinoma. High levels of SLX, CA19-9, SPan-1 and TA-4 were found in the serum of the original patient, but OCUG-1 cells produced only TA-4. We speculate that OCUG-1 may be a transitional form from adenocarcinoma to squamous cell carcinoma. Since OCUG-1 produces a high level of TA-4, it will be useful for the study of the biological nature of this carcinoma and the relationship between the expression of TA-4 and squamatization.

We investigated the association of urokinase-type plasminogen activator (u-PA) with tumor cell invasion and hepatic metastasis using human pancreatic cancer cell lines (SW1990, PANC-1, and RWP-1). We also examined the effect of the protease inhibitor, gabexate mesilate. SW1990 cells showed a higher u-PA activity (41.2 U/ml) in substrate assay and invasiveness (21.6% IV) than PANC-1 (14.3 U/ml, 10.3% IV) and RWP-1 (22.1, 13.5), which correlated with in vivo hepatic metastasis. Gabexate mesilate significantly reduced the u-PA activity, invasiveness, and hepatic metastasis of SW1990 cells. These findings suggest the possible application of protease inhibitors to prevent tumor invasion and metastasis.

The expression of the glycosphingolipid antigens was investigated in human colorectal carcinomas and adjacent normal tissues obtained by surgical operation from six patients. The glycosphingolipids, including both non-acidic glycolipid and ganglioside, were extracted from the tissue specimens and separated by two-dimensional thin-layer chromatography of silica gel. A novel neutral glycolipid antigen migrating lower than Le(a) blood group antigen was detected in the cancerous tissues of all six patients, but not in normal regions in the same patients. The structure of the glycosphingolipid was partially characterized by gas chromatography and gas chromatography/mass spectrometry of alditol acetate and negative ion fast atom bombardment mass spectrometry (FAB/MS) of the intact glycolipid, and TLC-immunostaining with monoclonal anti-Le(a), -Le(b), -sialyl Le(a), and -Le(Y) antibody, respectively. The novel neutral glycosphingolipid in colorectal cancer was identified to be the III(4)IV(2)Fuc(2)Lc(4)Cer, Le(b) antigen of the human Lewis blood system. Direct FAB/MS showed the ceramide was composed mainly of 2-hydroxy tetradecanon and C18 phytosphingosine. In such individuals, the expression of Le(b) antigen in normal tissues is absent or minimal. Based on this study, we suggest that this Le(b) antigen may be a human distal colorectal cancer-associated antigen, which will be helpful for diagnosis and treatment of distal colorectal cancer.

The relationship between cisplatin (cDDP) resistance and the expression of the molecular chaperonins hsp70, 60 and 27 was studied in two head and neck squamous cell carcinoma cell lines (UM-SCC-5 and UM-SCC-10B). The cDDP resistant variants were obtained by continuous exposure to escalating doses of cDDP. The IC(50)s of the parent and resistant variants (15th selection) of the UM-SCC-5 and UM-SCC-10b were 7.2+/-1.5 mu M and 20.1+/-1.2 mu M, and 6.5+/-0.6 mu M and 40.1+/-1.2 mu M, respectively. The emergence of cDDP resistance was closely related to the increase in basal expression of hsp60 (r=0.85 and 0.91 for UM-SCC-5 and UM-SCC-10B, respectively) resulting in a 2 to 3-fold increase in hsp60 mRNA in the cDDP resistant variants (15th selection). Expression of hsp27 was increased at higher levels of resistance in the UM-SCC-10B variants only (3 to 5-fold), and not in the UM-SCC-5 variants. In contrast to hsp60 and hsp27, the emergence of cDDP resistance did not lead to higher hsp70 mRNA levels. In addition, we determined the ability of cDDP resistant variants to induce the hsps 27, 60 and 70 in response to cDDP treatment. In the parental cell lines, hsp27 and hsp60 were all slightly increased after cDDP treatment (IC70 dose). However, only hsp60 could be induced in the resistant variants. In summary, emergence of cDDP resistance is associated with increased levels of hsp60 mRNA in the resistant variants and an inhibition of the transcriptional activity of hsp27 and hsp70.

The modifying effects of thymosin fraction 5 (TF5), a dialyzable glycoprotein extract obtained from calf thymus, on second stage urinary tract carcinogenesis after N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) initiation were investigated in NON/Shi male mice. BBN was administered for 8 weeks and then TF5 was administered subcutaneously twice a week for 12 weeks. The incidence of urinary bladder carcinomas was decreased in the TF5 treatment group, but the malignancy of the renal pelvic carcinomas appeared to be enhanced. We repeated the experiment to obtain confirmatory evidence, but found no significant effect, although TF5 did stimulate the immune surveillance system. The reason why this discrepancy occurred might be related to the degree of hydronephrosis or the confounding influence of renal pelvic carcinomas.

During the past 15 years, we encountered 1051 facial fractures. The number of patients with facial fractures accounted for 0.76% of the over all outpatients at the ENT clinic of Osaka City University Hospital. There were 496 nasal bone fractures, 111 maxillary fractures, 167 zygomatic fractures, 122 mandibular fractures, and 130 orbital fractures. These types of fractures accounted for 47.2%, 10.6%, 15.9%, 11.6%, and 12.4% of the over or facial fractures, respectively. The ratio of men to women was nearly three to one, with 794 men and 257 women. Among nasal fractures, injury causes were ranked in the order of fighting, sports, and traffic accident. Among facial fractures, injury causes were ranked in the order of the traffic accident, fighting, and falling down.

A murine monoclonal antibody, designated Nd2, was generated using a purified mucin from the human pancreatic cancer cell line SW1990 as the immunogen. Administration of Nd2 into the patients with pancreatic cancer has shown that Nd2 is of clinical use for in vivo diagnostics of pancreatic cancer. In this study, starting with a previously established hybridoma producing Nd2, we have established a transfected cell line expressing a recombinant chimeric Nd2 composed of mouse-derived antigen-binding variable regions and human-derived constant regions. The chimeric Nd2 was shown to bind purified mucin from SW1990 cells with the same dose-response curve as the original Nd2. Immunohistochemical experiments have shown that the reactivity of the chimeric Nd2 in normal and malignant pancreatic tissue is also the same as that of the original Nd2. Replacement of a substantial portion of the mouse protein makes this chimeric Nd2 more promising for clinical application than the original mouse-derived Nd2 because its antigenicity is expected to be reduced.

Development of resistance to cisplatin (cDDP) is a major obstacle in the cure of many cancers. Recently, a cDNA from cDDP-resistant human ovarian carcinoma cells was identified as the mitochondrial hsp60 chaperonin. The aim of this study was to determine the changes in expression of hsp60 during selection for cDDP resistance in two head and neck cancer cell lines, UMSCC5 and UMSCC10b and whether the emergence of resistance could be correlated with the level of hsp60 expression. We have selected cDDP-resistant variants of two squamous cell carcinoma cell lines, the UMSCC5 and UMSCC10b with levels of resistance varying from 1.5 to 6-fold. Concomitant with the emergence of resistance, the basal level of hsp60 increased 2 to 3-fold. In addition, less cDDP resistance as well as lower hsp60 levels were detected in cDDP resistant variants of the UMSCC10b cell line when selected in the presence of tamoxifen, suggesting a correlation between the intrinsic level of hsp60 expression and cDDP resistance. Using linear regression analysis both UMSCC5 and UMSCC10b cell lines demonstrated a high degree of correlation with coefficients of 0.91 and 0.90, respectively. In conclusion, the expression of hsp60 was closely related to the development of cDDP resistance and could be used as a marker for the emergence of cDDP resistance.

Systemic chemotherapy has been widely used for the purpose of inducing remission of advanced cancer of the cervix. Almost all anti-cancer drugs have been applied to this way of chemotherapy, however, the high effectiveness of cisplatin is worthy of notice, and this drug plays a central role in combination chemotherapy. Local hyperthermia, drug-induced hypertension and intra-arterial infusion of anti-cancer drugs are the representative methods to reinforce the effect of chemotherapy. Adjuvant chemotherapy has to be designed to adapt the extremely slow growing potential of residual microcarcinomas after surgery. Oral administration of tegafur for two years is a choice of maintenance chemotherapy preventive against postoperative recurrence.

Reported is the case of a 71 year-old male with a poorly differentiated adenocarcinoma of the colon that had invaded the submucosal layer. A barium enema had revealed a polyp, 1.5 cm in diameter, in the descending colon, and an endoscopic polypectomy was performed. The resected specimen mainly consisted of an adenoma and a partial, poorly differentiated adenocarcinoma that had massively infiltrated into the submucosal layer. A left hemicolectomy was carried out, and no carcinoma was identified in the resected colon and/or the regional lymph nodes.

A case of secondary plasmacytoma (Ig. D. lambda) of the breast, associated with multiple myeloma, is reported, involving a 60-year-old woman in whom multiple tumors of the right breast appeared during the course of her treatment for multiple myeloma. These breast tumors were firm, non-tender, clear-margined, smooth-surfaced, and showed good mobility. An ultrasonogram revealed that the tumors had the shape of a clear-margined anechoic mass like a cyst. A histological diagnosis of a biopsied specimen determined plasmacytoma (Ig. D, lambda) and the patient was treated with chemotherapy which caused the tumors to greatly reduce.