YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes.

OBJECTIVE: YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. SUBJECTS AND METHODS: The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. RESULTS: YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. CONCLUSIONS: GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis.

Relativistic calculation of nuclear magnetic shielding tensor using the regular approximation to the normalized elimination of the small component. III. Introduction of gauge-including atomic orbitals and a finite-size nuclear model.

The relativistic calculation of nuclear magnetic shielding tensors in hydrogen halides is performed using the second-order regular approximation to the normalized elimination of the small component (SORA-NESC) method with the inclusion of the perturbation terms from the metric operator. This computational scheme is denoted as SORA-Met. The SORA-Met calculation yields anisotropies, Delta sigma = sigma(parallel) - sigma(perpendicular), for the halogen nuclei in hydrogen halides that are too small. In the NESC theory, the small component of the spinor is combined to the large component via the operator sigma x piU/2c, in which pi = p + A, U is a nonunitary transformation operator, and c approximately = 137.036 a.u. is the velocity of light. The operator U depends on the vector potential A (i.e., the magnetic perturbations in the system) with the leading order c(-2) and the magnetic perturbation terms of U contribute to the Hamiltonian and metric operators of the system in the leading order c(-4). It is shown that the small Delta sigma for halogen nuclei found in our previous studies is related to the neglect of the U(0,1) perturbation operator of U, which is independent of the external magnetic field and of the first order with respect to the nuclear magnetic dipole moment. Introduction of gauge-including atomic orbitals and a finite-size nuclear model is also discussed.

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity.

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

Effect of substrate rigidity in tissue culture on the function of insulin-secreting INS-1E cells.

Insulin-secreting INS-1E cells are a useful tool in diabetes research. However, during permanent culture the cells tend to lose their ß cell phenotype, with resultant loss of insulin-secretory responsiveness. This can be at least partially attributed to inappropriate cell culture conditions. One of the important causative factors is the rigidity of the extracellular matrix. We have therefore systematically studied the performance of INS-1E insulin-secreting cells cultured on polyacrylamide gels of different stiffnesses and analysed changes in insulin content and secretion, glucokinase enzyme activity, gene expression of ß cell transcription factors and cell death and proliferation rates. INS-1E cells were cultured on polyacrylamide gels with a wide range of rigidities, including the one that simulates the stiffness of the pancreas. We detected changes in insulin content and the insulin-secretory response to glucose stimulation in parallel to the increasing stiffness of the polyacrylamide gels in the range 1700-111 000 Pa. On substrates with the highest and lowest rigidities, 322 and 111 000 Pa, the cells mainly formed pseudo-islets, while at rigidities of 1700-64800 Pa, including the rigidity of native pancreas tissue (3100 Pa), cells grew as a monolayer attached to the polyacrylamide gel surface. These observations provide evidence for an apparent mechanosensitivity of insulin-secreting INS-1E cells affecting morphology and cellular functions. The results can also provide practical advice regarding a selection of the materials appropriate for successful cell culture of insulin-secreting cells. Copyright © 2014 John Wiley & Sons, Ltd.

Palmitoylation of the canine histamine H2 receptor occurs at Cys(305) and is important for cell surface targeting.

To determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys(305) to Ala (A(305) receptor) mutation was generated. Wild-type (WT) and A(305) receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A(305), receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys(305). Immunocytochemistry of WT and A(305) receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A(305) receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A(305) receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A(305) receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10(-5) M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A(305) receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A(305) receptors were incubated with 10(-5) M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A(305) receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A(305) receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability.

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats.

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.

No correlation of plasma cell 1 overexpression with insulin resistance in diabetic rats and 3T3-L1 adipocytes.

Membrane glycoprotein plasma cell 1 (PC-1) has been shown to be increased in type 2 diabetes and involved in insulin resistance through inhibiting the insulin receptor tyrosine kinase, which was demonstrated using cultured breast cancer cells. However, other reports have shown contradictory results in Chinese hamster ovary cells and in vitro kinase assay. Thus, we considered it necessary to investigate the effect of PC-1 using highly insulin-sensitive cells. Here, we used two of the following approaches: 1) investigating PC-1 expression levels in insulin-responsive tissues in rat models of diabetes and 2) overexpressing PC-1 in 3T3-L1 adipocytes. We found that PC-1 was highly expressed in insulin-responsive tissues, such as liver and adipose tissue, in normal rats. However, high-fat feeding or streptozotocin-induced diabetes did not change its expression levels in liver, adipose tissue, and skeletal muscle. Thus, PC-1 expression levels were not associated with high-fat-diet-induced insulin resistance or hyperglycemia. Although PC-1 was increased in adipose tissue in Zucker fatty rats (protein level, by 50%; mRNA level, by 90%), its expression levels in liver and skeletal muscle, tissues that are more responsible for whole body glucose metabolism than adipose tissue, did not significantly differ from those in normal rats. Next, we overexpressed PC-1 in 3T3-L1 adipocytes using an adenovirus transfection system. PC-1 expression was markedly increased to a level 16-fold greater than that in normal human adipose tissue, which is higher than the previously reported levels in diabetic patients. However, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1, activation of phosphatidylinositol 3-kinase, and glucose uptake were not affected by PC-1 overexpression. These results strongly suggest that increased PC-1 expression is not causally related to insulin resistance.

Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction.

Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[3H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110alpha, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation.

Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats.

To elucidate the mechanism of obesity-related insulin resistance, we investigated the impaired steps in the processes of phosphatidylinositol (PI) 3-kinase activation through binding with insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in liver and muscle of Zucker fatty rats. The expressions of IRS-1 and IRS-2 were shown to be downregulated in both liver and muscle in fatty rats (hepatic IRS-1, 83%; hepatic IRS-2, 45%; muscle IRS-1, 60%; muscle IRS-2, 78%), resulting in decreased tyrosine phosphorylation in response to insulin stimulation. Despite the decrease in the tyrosine phosphorylation levels of hepatic IRS-1 and IRS-2 being mild to moderate, associated PI 3-kinase activities were dramatically decreased in fatty rats (IRS-1, 14%; IRS-2, 10%), which may suggest alteration in the sites of phosphorylated tyrosine residues of hepatic IRS-1 and IRS-2. In addition, we demonstrated that the expressions of p85alpha and p55alpha regulatory subunits of PI 3-kinase were reduced (p85alpha, 67%; p55alpha, 54%), and that the p50alpha regulatory subunit was markedly upregulated (176%) in the livers of fatty rats without apparent alterations in expressions of the catalytic subunits p110alpha and p110beta. These alterations may reflect the obesity-related insulin resistance commonly observed in human NIDDM.

Structure and function of phosphatidylinositol-3,4 kinase.

Activation of phosphatidylinositol (PI)-kinase is involved in the regulation of a wide array of cellular activities. The enzyme exists as a dimer, consisting of a catalytic and a regulatory subunit. Five isoforms of the regulatory subunit have been identified and classified into three groups comprising respectively 85-kDa, 55-kDa, and 50-kDa proteins. Structural differences in the N-terminal regions of the different group members contribute to defining their binding specificity, their subcellular distributions, and their capacity to activate the 110-kDa catalytic subunit. Two widely distributed isoforms of the catalytic subunit have been identified-p110alpha and p110beta. Despite the fact that they bind to the p85alpha regulatory subunit similarly, p110alpha and p110beta appear to have separate functions within cells and to be activated by different stimuli. Moreover, although p85/p110 PI-kinase almost exclusively phosphorylates the D-3 position of the inositol ring in phosphoinositides when purified PI is used as a substrate in vitro, it appears to phosphorylate the D-4 position with similar or higher efficiency in vivo. Thus, it is highly probable that p85/p110 PI-kinase transmits signals to downstream targets via both D-3- and D-4-phosphorylated phosphoinositides.

Regulation of phosphoinositide metabolism, Akt phosphorylation, and glucose transport by PTEN (phosphatase and tensin homolog deleted on chromosome 10) in 3T3-L1 adipocytes.

To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinositide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wild-type PTEN and its phosphatase-dead mutant (C124S) with or without an N-terminal myristoylation tag were overexpressed in Sf-9 cells and 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110alpha catalytic subunit of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the accumulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate induced by p110alpha. In contrast, overexpression of the C124S mutants apparently increased these accumulations. In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.

Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells.

A histamine H2 receptor, which had been mutated at its glycosylation site and tagged at its N-terminus with an HA tag (HA-H2 receptor), was expressed in Sf9 cells and COS7 cells. Immunoprecipitation and immunoblotting of HA-H2 receptors with alphaHA antibody revealed four bands of 31.5 +/- 2.5 kDa, 59.0 +/- 6.0 kDa, 80.5 +/- 4.5 kDa and 120 kDa. These bands were also detected by immunoblot using anti-H2 receptor serum (C-terminus). In addition, H2 receptors without the HA-tag coimmunoprecipitated with HA-tagged H2 receptors devoid of the 51 C-terminal amino acids, via immunoprecipitation with alphaHA antibody, when the two receptors were coexpressed. These results suggest that H2 receptors are present as receptor oligomers and that the C-terminal portion is not involved in the formation of these oligomers.

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations.

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.

Imidapril, an angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and improving vascular resistance in the Zucker fatty rat.

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive agents, that inhibit the conversion of angiotensin I to angiotensin II, resulting in smooth-muscle relaxation and a reduction of vascular resistance. Recently, it has been suggested that ACE inhibitors improve insulin resistance in diabetic patients. To investigate the effect of an ACE inhibitor on insulin sensitivity, insulin signaling, and circulation, imidapril was administered orally or intraduodenally to Zucker fatty rats. Oral administration of imidapril improved insulin sensitivity based on the results of an oral glucose tolerance test (OGTT) and a decrease in urinary glucose secretion. Phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with hepatic insulin receptor substrate-1 (IRS-1) in the insulin-stimulated condition was significantly enhanced 110% without a significant alteration in tyrosine phosphorylation of IRS-1 in the imidapril-treated group. In muscle, IRS-1 tyrosine phosphorylation and PI 3-kinase activity associated with IRS-1 in the insulin-stimulated condition were enhanced 70% and 20%, respectively, in the imidapril-treated group. In contrast, an alteration of the IRS-2 pathway was observed only in liver; a significant insulin-induced increase in the IRS-2-associated PI 3-kinase over the basal level was observed in the imidapril-treated group but not in the control. In addition, treatment with imidapril was shown to significantly reduce blood pressure and increase blood flow in the liver and muscle. These results suggest that the ACE inhibitor imidapril may improve insulin sensitivity not only by acting directly on the insulin signaling pathway but also by increasing blood flow in tissues via normalization of vascular resistance, a major cause of hypertension.

Biochemical modulation of 5-fluorouracil with a streptococcal preparation, OK-432, against murine colon-26 carcinoma.

Conventional therapy for colorectal carcinoma using 5-fluorouracil (5-FU) has shown limited antitumor action. The purpose of our study was to investigate synergistic antitumor effects of the streptococcal preparation of OK-432 and 5-FU, and to elucidate the mechanisms of interaction between the 2 agents in mice. Biochemical modulation of OK-432 and 5-FU were determined in vivo against colon-26 carcinoma. The concentration of 5-FU and its metabolites, and the activity of thymidylate synthase and thymidine kinase, respectively, were measured using cytosolic extracts of the tumors. Combination treatment with OK-432 produced a significant increase in intratumor 5-FU and 5-FU in RNA (F-RNA) concentrations, increased the thymidylate synthetase inhibition rate, and decreased thymidine kinase activity, as compared with the results observed in the control mice. These additive antitumor effects are obtained by use of the 2 agents; the mechanism of action is considered to be the suppression of both the de novo and the salvage pathway for DNA synthesis, along with the suppression of RNA synthesis.

Trichilemmal carcinoma on the thigh: report of a case.

A 79-year-old Japanese man with a trichilemmal carcinoma (TC) on the right thigh is described. To the best of our knowledge, he is the fifth patient with a TC on the thigh reported from Japan.

Angiosarcoma which developed on the abdominal wall: report of a case.

We report a 68-year-old Japanese woman with angiosarcoma (AS) on the abdominal wall. To the best of our knowledge, she is the third such patient reported from Japan.

Prurigo nodularis associated with advanced gastric cancer: report of a case.

In a male Japanese patient, prurigo nodularis (PN) appeared in association with gastric cancer. The cutaneous pruriginous lesions dramatically improved soon after total gastrectomy without any treatment for the skin lesions. Peripheral eosinophilia seen before the operation also rapidly disappeared. These data suggest that some cytokines involved in gastric cancer might have played an important role in the development of PN in our patient.

Autologous serum deprivation restored IL-1 receptor antagonist production by peripheral blood mononuclear cells in patients with gastric cancer.

It has been established that cancer patients have immunosuppressive substances in their sera that depress cellular immunity. Although plasma exchanges have been attempted to remove these substances and to improve immunity to cancer, little is known about its mechanism from the viewpoint of cytokine pattern. The levels of the cytokines, tumour necrosis factor-alpha, interleukin 1beta, interleukin 6, interferon-gamma and interleukin-1 receptor antagonist (IL-1ra) by peripheral blood mononuclear cells (PBMC) were determined simultaneously by the whole-blood assay and the PBMC assay in 20 patients with gastric cancer and in 10 healthy volunteers. In both assays the cytokine levels were lower in patients with cancer compared with healthy controls, with the exception of IL-1ra. In the PBMC assay, the IL-1ra level in cancer patients was significantly higher than that in controls. No statistical correlation between the cytokine levels determined by the two assays was found. We suggest that autologous serum deprivation restored and enhanced IL-1ra production, and normalized the cytokine cascade in immune response, in patients with gastric cancer.

Schedule-dependent combined sensitivity testing of anti-cancer agents in human gastric carcinoma cell lines.

The efficacy of combination chemotherapy for gastric carcinoma has been unsatisfactory, although the prognosis of advanced and recurrent disease has improved with the introduction of cisplatin (CDDP). This study examines the effect of the anti-cancer therapies CDDP, doxorubicin (ADM) and etoposide (VP-16) on the cell cycle and their cytotoxicity against two gastric carcinoma cell lines: MKN-28 (well differentiated) and MKN-45 (poorly differentiated). The treatments have different cytocidal mechanisms, and they were studied in dual combinations. For all combinations studied, cytotoxicity against MKN-45 was higher than against MKN-28. For ADM plus CDDP, or ADM plus VP-16, cytotoxicity was higher in patients pretreated with ADM than other regimens. The highest anti-tumour activity against both cell lines was obtained with ADM followed by CDDP (we have obtained good clinical results with this regimen). Schedule-dependent combined sensitivity testing of anti-cancer agents will be useful for the clinical application of therapies.