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The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is expressed at higher levels in a wide variety of solid tumors compared to adjacent normal tissues. Patients with PD-ECGF/TP-positive colon and esophageal tumors have a poorer prognosis than those with negative tumors. The expression of PD-ECGF/TP is a prognostic factor independent of microvessel density suggesting that TP has effects on tumor progression independent of its angiogenic activity. Evidence that hypoxia and apoptosis affect tumor growth prompted us to determine whether increased expression of PD-ECGF/TP prevents apoptosis induced by hypoxia. KB/TP cells transfected with a PD-ECGF/TP cDNA were resistant to hypoxia-induced apoptosis. Among the degradation products of thymidine produced by PD-ECGF/TP, 2-deoxy-D-ribose and thymine partially prevented hypoxia-induced apoptosis. The ability of 1 microM 2-deoxy-D-ribose in combination with the same concentration of thymine to prevent hypoxia-induced apoptosis was similar to that of the overexpressed TP in KB cells. A concentration of 1 microM 2-deoxy-L-ribose abrogated the effects of these degradation products of thymidine. These findings suggested that TP can confer resistance to apoptosis induced by hypoxia and the degradation products of thymidine are involved in this resistance. Expression of PD-ECGF/TP may play an important role in the progression of solid tumors, and inhibitors of TP and analogs of the degradation products of thymidine may suppress the growth of tumors by promoting apoptosis.

The leukotriene D4 (LTD4) receptor antagonist, 4-oxo-8-[p-(4-phenylbutyloxy)benzoylamino]-2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate (ONO-1078) is used for the treatment of allergic asthma and other immediate hypersensitivity diseases. We examined the effect of ONO-1078 on the sensitivity to vincristine (VCR) of MRP overexpressing multidrug-resistant CV60 and its parental drug-sensitive KB-3-1 cell lines. The sensitivity to VCR of KB-3-1 and CV60 cells was increased 13- and 15-fold, respectively, by ONO-1078 at the maximum non-toxic concentration (100 microM). The VCR sensitivity of multidrug-resistant KB-C2 cells that overexpressed P-gp was increased 2.6-fold by ONO-1078. The accumulation of VCR in KB-3-1, CV60 and KB-C2 cells was significantly increased by ONO-1078. The efflux of VCR from KB-3-1 cells was not inhibited, but that from CV60 cells was enhanced compared with that from KB-3-1 cells and was partially inhibited by ONO-1078. ONO-1078 competitively inhibited the ATP-dependent [3H]LTC4 uptake in membrane vesicles isolated from CV60 cells. These findings suggest that ONO-1078 inhibits the transporting activity of MRP and that ONO-1078 increases the sensitivity to VCR of KB-3-1 cells by increasing the VCR uptake in the cells.

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor and has angiogenic activity. In this study, we investigated the expression of dThdPase in ductal adenocarcinoma of the pancreas and examined the correlation between dThdPase expression and clinicopathological factors and clinical outcome. dThdPase expression was demonstrated by immunohistochemistry in the cytoplasm of tumor cells in 59% of the 54 patients studied. The expression of dThdPase correlated significantly with a poor prognosis (P=0.013). Significant correlations were also observed between dThdPase expression and extrapancreatic neural plexus invasion and the presence of postoperative hepatic metastases (P=0.05 and 0.03, respectively). The average microvessel count in dThdPase-positive tumors was significantly higher than that in dThdPase-negative tumors (P < 0.0001). These findings suggest that dThdPase expression in pancreatic adenocarcinoma enhances the abilities of tumor invasion and/or metastasis through its angiogenic properties.

We retrospectively analyzed the prognostic significance of angiogenesis and the relationships between tumor angiogenesis and clinopathological variables in a series of 127 patients with primary esophageal squamous cell carcinoma which were curatively resected. Vessels were stained with anti-factor VIII polyclonal antibody and areas with the highest number of microvessels were counted in a x400 field. Microvessel counts were significantly correlated with pN category, pM category, venous invasion and recurrence (p=0.002, p=0.040, p=0.016 and p=0.0013, respectively). The proportion of patients with recurrence increased in proportion to the number of microvessels. multivariate analysis using Cox's proportional hazard modelling showed angiogenesis assessed by microvessel count affected the poorer prognosis of patients with esophageal squamous cell carcinoma (hazard ratio, 1.03; 95%CI, 1.00-1.07), although it was not a significant independent prognostic factor (P=0.088). This study suggest that angiogenesis assessed by microvessel count is a marker for relapse and prognosis in patients with esophageal squamous cell carcinoma.

Background: Multidrug resistance protein (MRP) is a membrane phosphoglycoprotein with an Mr of 190,000 that is involved in the non-P-glycoprotein mediated multidrug resistance of human tumor cells. The aim of this study was to determine the clinicopathologic relevance of MRP expression in human gastrointestinal tract carcinomas., Methods: The authors prepared a rabbit antiserum against MRP that does not cross-react with P-glycoprotein and retrospectively examined the expression of MRP in 86 squamous cell carcinomas of the esophagus, 103 adenocarcinomas of the stomach, and 139 colorectal adenocarcinomas by immunohistochemistry. None of the patients in this study had received prior chemotherapy., Results: The proportion of MRP positive samples in the squamous cell carcinomas of the esophagus (62.8%, 54 of 86) was significantly higher than that in the adenocarcinomas of the stomach (34.1%, 35 of 103) and the colorectal adenocarcinomas (40.3%, 56 of 139) (P <0.01). The proportion of MRP positivity in the well-differentiated carcinomas was significantly higher than that in moderately or poorly differentiated carcinomas. MRP expression was independent of gender, lymph node metastasis, and tumor progression., Conclusions: These data indicate that the expression of MRP is correlated with the differentiation of carcinoma cells in the gastrointestinal tract and may be involved in the intrinsic drug resistance of well-differentiated squamous cell carcinoma of the esophagus.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in various types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We measured the dThdPase activity and the level of thrombomodulin (TM), as a marker for endothelial cells, in ovarian carcinomas, benign tumors and normal ovarian tissues. The average dThdPase activity of ovarian carcinomas (10.86+/-6.98 nmol/100 mu g protein/h) was significantly higher than that of benign tumors (4.66+/-3.91 nmol/100 mu g protein/h) or normal tissues (2.52+/-1.90 nmol/100 mu g protein/h). The expression of dThdPase detected by immunoblot analysis was well correlated with dThdPase activity. In an immunohistochemical study, the expression of dThdPase was more frequently observed in ovarian carcinomas than in benign tumors or normal tissues. dThdPase activity in human ovarian carcinomas was significantly correlated with the expression of TM in human ovarian carcinomas. These findings suggest that dThdPase expression is significantly correlated with angiogenesis in ovarian tumors.

Purpose: Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. We examined whether TP expression in renal cell carcinoma (RCC) is associated with microvessel density as a marker of angiogenesis, clinicopathologic characteristics, and outcome., Patients and Methods: The enzymatic activity and expression of TP were examined in 18 RCCs and 19 kidney tissues not grossly involved with tumor from 24 patients with 13 paired samples and 11 unpaired samples by spectrophotometry and immunoblotting. The relationship between TP expression and microvessel density was assessed by immunohistochemistry in 133 RCCs., Results: The median enzymatic activity of TP in RCCs was nine fold higher than that in nonneoplastic kidney tissues (P < .001). Similar results were obtained by immunoblot analysis. According to the TP staining profile, tumors were classified as no or low, intermediate, or high TP-expressing tumors. TP positivity was significantly correlated with microvessel density. TP expression was correlated with tumor grade, but there was no significant association between TP expression and other clinicopathologic characteristics. TP expression as a prognostic variable was studied using Cox's proportional hazards model. TP overexpression was an independent prognostic factor (hazards ratio, 3.95; 95% confidence interval, 0.98 to 15.89; P = .039) as were nodal category, metastases category, tumor grade, and venous invasion., Conclusion: These findings suggest that TP expression is correlated with microvessel density in RCC and is an unfavorable independent prognostic factor. The future development and characterization of TP inhibitors may provide a novel approach to the therapy of RCC.

The production of both nitric oxide (NO) and superoxide increases in septic shock. The cogeneration of these molecules is known to yield peroxynitrite, which preferentially nitrates tyrosine residues of protein and non-protein origins. We present evidence of peroxynitrite production in septic shock by measuring plasma nitrotyrosine. The nitrotyrosine was measured by an HPLC C-18 reverse-phase column and ultraviolet detector in chronic renal failure patients with or without septic shock, and in healthy volunteers. Plasma nitrite + nitrate (NOx) was also measured to evaluate NO production. Nitrotyrosine was selected as an index for production of peroxynitrite because the direct measurement of peroxynitrite in vivo is difficult. Patients with renal failure were selected in order to minimize nitrotyrosine excretion through the kidney. Plasma nitrotyrosine levels were not detectable in volunteers, 28.0 +/- 12.3 microM (1.6 +/- 1.1% of total tyrosine) in renal failure patients without septic shock, and 118.2 +/- 22.0 microM (5.5 +/- 1.2% of total tyrosine) in patients with septic shock. NOx levels were also higher in patients with septic shock than in patients without septic shock (173.9 +/- 104.7 vs. 75.6 +/- 19.1 microM). Although renal failure itself increases plasma concentrations of both molecules, the higher levels in patients with septic shock suggest that peroxynitrite is generated and the nitration of tyrosine residues is increased in this disease.

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We examined the involvement of TP activity in tumor growth and angiogenesis. KB cells were transfected with wild-type or mutant (L148R) PD-ECGF cDNA, and two sublines with high TP activity, KB/wt4 and KB/wt6, and one subline with no TP activity, KB/L148R, were cloned, respectively. The doubling times of these subclones in vitro were similar to that of KB cells. However, the growth of KB/wt4 and KB/wt6 cells was significantly faster when xenografted into nude mice than that of control cells with no TP activity. The tumors with high TP activity (KB/wt4 and KB/wt6) had significantly more microvessels than those with no TP activity (KB/-, KB/CV and KB/L148R) (P<0.01). These results, taken together with previous reports, suggest that the TP enzyme activity itself is involved in angiogenesis and growth of the KB tumors.

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.

An active efflux pump for cis-diamminedichloroplatinum(II) (cisplatin) has been identified in cisplatin-resistant KCP-4 cells isolated from human epidermoid carcinoma KB-3-1 cells. The adenosine triphosphate(ATP)-dependent transport of leukotriene C4 (LTC4), an endogenous substrate for the glutathione S-conjugate export pump(GS-X pump), has been found in membrane vesicles prepared from KCP-4 cells. Multidrug resistance-associated protein (MRP) has also been identified as an ATP-dependent LTC4 transporter. To examine whether the GS-X pump expressed in KCP-4 cells in MRP, we investigated the expression of MRP in KCP-4 cells and compared the LTC4 transporting activity of GS-X pump expressed in KCP-4 cells with that of MRP. The level of MRP gene expression in KCP-4 cells was low and similar to that in KB-3-1 cells. MRP was not detected in membrane vesicles prepared from KB-3-1 and KCP-4 cells by immunoblot analysis. The ATP-dependent transport of LTC4 in KCP-4 and C-A120 vesicles showed saturable kinetics with an apparent Km of 0.18 microM and 0.25 microM, respectively. [3H]LTC4 transport in KCP-4 vesicles was more inhibited by 2,4-dinitrophenyl-S-glutathione(DNP-SG), bis-(glutathionato)-platinum(II) (GS-platinum) complex and glutathione disulfide(GS-SG) and less by LTD4 compared with that in C-A120 vesicles. The character of the LTC4 transporter expressed in KCP-4 vesicles is similar but not identical to that of MRP. Our results suggest that a glutathione S-conjugate export pump which is different from MRP exists in cisplatin-resistant KCP-4 cells.

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We previously found that thymidine phosphorylase (dThdPase) and PD-ECGF are the same protein., Purpose: We retrospectively examined the expression of dThdPase in primary colorectal carcinomas, its association with angiogenesis and clinicopathologic findings, and its prognostic value., Methods: Tissues were obtained from the tumors of 163 patients whose colorectal carcinomas were completely removed by surgery. Microvessels assessed by immunostaining endothelial cells for factor VIII were counted on a 400x field in the most active areas of neovascularization within the tumor. We purified the monoclonal antibody against dThdPase and studied the expression of dThdPase in the same serial sections used for the detection of factor VIII. Those who carried out microvessel counting and dThdPase expression assessment had no knowledge of clinicopathologic findings. The significance of dThdPase in the prognosis of patients with colorectal carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1984 through 1991. Reported P values are from two-sided tests of statistical significance., Results: The mean microvessel count (+/- standard deviation) in dThdPase-positive colorectal carcinoma specimens (17.5 +/- 7.2) was higher (P < .001) than that in dThdPase-negative carcinoma specimens (9.3 +/- 5.5). The dThdPase positivity was in accordance with the microvessel count. dThdPase positivity showed highly significant statistical associations with tumor size, extent of invasion, lymph node metastasis, lymphatic invasion, and venous invasion. Cox regression analysis revealed that dThdPase expression was prognostic for poor disease outcome after adjustment for Dukes' stage and microvessel count., Conclusions: These findings suggest that higher levels of dThdPase expression in colorectal carcinomas are associated with more extensive angiogenesis, poor clinical and laboratory findings, and unfavorable clinical outcome., Implications: Inhibition of dThdPase in human colorectal carcinomas might improve prognosis for some patients.

YM-24074 (formerly called YL-01869P) is a new antibiotic and type I collagenase inhibitor. The structure of this compound was elucidated by spectroscopic analysis and confirmed by acid degradation to be N-(1"-acetyl-3"-methylbutyl)-2- [2'-[(N-hydroxycarbamoyl)methyl]heptanoyl]-hexahydropyrid azine-3-carboxamide. The stereochemistry of two of three chiral centers was determined by degradation products to be 3S, 2'R.

Background: Angiogenesis is reportedly correlated with metastasis, relapse, and prognosis in some types of tumors. Hematogenous or lymph node metastasis and local recurrence are the main elements related to the death of patients with colorectal carcinoma. Thus, the authors examined the microvessel count in colorectal carcinoma to determine how angiogenesis correlates with clinicopathologic factors and prognosis., Methods: Paraffin embedded sections from 166 patients with primary colorectal carcinomas that had been completely removed were analyzed for angiogenesis. Vessels were stained with anti-factor VIII polyclonal antibody, and areas with the most discrete microvessels were counted in a 400x field., Results: Tumor size was significantly correlated with microvessel count. Microvessel counts from patients with lymph node metastasis, lymphatic vessel invasion, venous vessel invasion, or relapse were significantly higher than those without. Furthermore, microvessel count was an independent prognostic factor (P = 0.007), whereas the Dukes stage had more significant prognostic value (P < 0.001) according to the multivariate Cox hazard analysis., Conclusions: This study suggested that angiogenesis assessed by the microvessel count was a marker of relapse and prognosis of patients with colorectal carcinoma.

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. Since dThdPase seems to have an important role in angiogenesis of tumours, we measured the activity and expression of dThdPase in various tumours and the adjacent non-neoplastic tissues. We assayed dThdPase activity by spectrophotometric means, and the expression of dThdPase was examined by immunoblotting and by immunohistochemical staining using a monoclonal antibody against dThdPase. In the oesophagus, stomach, colorectum, pancreas, and lung, dThdPase activity in carcinomas was significantly higher (P < 0.05) than that in the adjacent non-neoplastic tissues. The expression level of dThdPase detected by immunoblotting correlated well with the activity of dThdPase. In the oesophagus, stomach, colorectum, gall bladder, pancreas and lung, the proportion of dThdPase-positive tumours was significantly higher (P < 0.05 or 0.01) than that of the dThdPase-positive adjacent normal tissues. In oesophageal, gastric colorectal and lung carcinomas, the proportion of dThdPase positivity in advanced carcinomas was significantly higher (P < 0.01) than that in early carcinomas. Tumour-infiltrative macrophages or lymphocytes in the lymph node, alveolar macrophages and Kupffer cells expressed high levels of dThdPase. The results indicate that dThdPase activity and expression level in many tumours are higher than those in the adjacent non-neoplastic tissues, and that dThdPase may have an important role in the proliferation of these solid tumours.

MRP has been identified as another multidrug-resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug-resistance KB sublines (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug-sensitive human KB cells. MRP gene expression was elevated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous-cell carcinomas of the lung.

The activity of thymidine phosphorylase (dThdPase) has been reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immunostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a x400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P < 0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P < 0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P < 0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P < 0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P < 0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P < 0.001) or lymph node metastasis (P = 0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase-negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.

Thymidine phosphorylase (dThdPase) is an angiogenic enzyme and seems to be related to an angiogenesis in human colorectal carcinoma. The incidence of dThdPase-positive cells was significantly correlated with microvessel count in 21 human colorectal carcinomas. Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), interferon alpha (IFN-alpha) interferon gamma (IFN-gamma) induce dThdPase activity in human cancer cell lines. To study whether this phenomenon occurs in the human colorectal carcinomas, we examined the correlation between dThdPase activity and the expression levels of IL-1 alpha, TNF-alpha, IFN-alpha and IFN-gamma in colorectal carcinoma tissues. dThdPase activity was assayed by the methods of Friedkin and Robert, and the expression level of IL-1 alpha, TNF-alpha, IFN-alpha and IFN-gamma was determined by ELISA. dThdPase activity was significantly correlated with the amount of IL-1 alpha (n = 19, r = 0.347, P = 0.0001), INF-alpha (n = 18, r = 0.717, P = 0.008), and IFN-gamma (n = 4, r = 0.9777, P = 0.0234) in human colorectal carcinomas. However, the dThdPase activity was not correlated with the amount of TNF-alpha (n = 21, r = 0.235, P = 0.2682). These results suggested that the expression levels of IL-1 alpha, IFN-alpha and IFN-gamma are correlated with dThdPase activity in human colorectal carcinomas and that these cytokines may cause angiogenesis by inducing the expression of dThdPase.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in several types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and that dThdPase has angiogenic activity. We measured dThdPase activity and the level of thrombomodulin (TM) as a marker for endothelial cells in colorectal carcinomas and adjacent normal tissues from 21 patients, and in adenomas from 13 patients. The average dThdPase activity of colorectal carcinomas (11.58 +/- 6.30 nmol/100 micrograms protein/h) was significantly higher than that of adenomas (8.57 +/- 4.14 nmol/100 micrograms protein/h) or normal tissues (4.89 +/- 3.16 nmol/100 micrograms protein/h). In immunohistochemical study, the expression of dThdPase was observed more frequently in colorectal carcinomas than in adenomas or normal mucosas. The amount of TM in colorectal carcinomas (8.32 +/- 5.07 ng/100 micrograms protein) was significantly higher than that of adenomas (4.51 +/- 4.49 ng/100 micrograms protein) or normal tissues (3.51 +/- 2.78 ng/100 micrograms protein). dThdPase activity in human colorectal carcinomas, adenomas and normal tissues was significantly correlated with the expression of TM in these tissues. These results indicate that the expression levels of both dThdPase and TM in colorectal carcinomas are higher than those in colorectal adenomas and normal tissues and suggest that dThdPase may be involved in angiogenesis in human colorectal carcinomas, adenomas and normal tissues.

Novel pepstatin A-sensitive Candida albicans aspartyl protease inhibitors, named YF-0200R-A and B, were isolated by column chromatography and preparative HPLC from the fermentation broth of Streptomyces sp. YF-0200R. The structures of YF-0200R-A and B were elucidated by spectroscopic analysis as alpha, beta and gamma, delta unsaturated fatty acids with two or three hydroxyl groups. YF-0200R-A and B inhibit aspartyl protease from Candida albicans with IC50 values of 6.5 x 10(-4) M and 6.2 x 10(-4) M, respectively.

A formulation of meropenem, a novel carbapenem antibiotic for injection, was developed as a vial filled with a mixture of meropenem and dried sodium carbonate. During the design phase, we studied the effect of water in the formulation on the stability of meropenem in the solid state. Meropenem is obtained as trihydrate, whose moisture content is 12.35% and is nonhygroscopic. Dehydrated meropenem, whose moisture content was 3.4%, took up moisture quickly even under low humidity (33% RH). Also, the chemical stability of dehydrated meropenem was poor compared with that of untreated meropenem, which is quite stable. Degradation of meropenem by free water was considered as a possible cause of the poor stability. Degradation of meropenem due to liberation of its crystal water to free water was also observed when meropenem was micronized by pneumatic pulverization. Crystal water of meropenem was found to stay bound and to be almost inert in the formulation. Thus, meropenem injection formulation is stable for long time at room temperature.

(+)-trans-4-(3-dodecanoyl-2,4,6-trihydroxyphenyl)-7-hydroxy-2-(4- hydroxyphenyl)chroman (YM-26567-1), a novel natural product isolated from the fruit of Horsfieldia amygdaline, dose-dependently inhibited group II phospholipase A2 (PLA2) prepared from rabbit platelet with an IC50 value of 6.7 microM (4.6-9.6 microM, n = 4). In contrast to irreversible PLA2 inhibitors such as manoalide and p-bromophenacyl bromide, the PLA2 inhibition of YM-26567-1 was independent of preincubation time. Lineweaver-Burk analysis revealed that YM-26567-1 behaved as a competitive inhibitor of rabbit platelet PLA2 with a Ki value of 1.6 +/- 0.3 microM (n = 5). Although YM-26567-1 also competitively inhibited group I PLA2 derived from porcine pancreas, the Ki value was approximately 10-fold greater for porcine pancreas than for rabbit platelet PLA2. In vivo, topical application of YM-26567-1 to the mouse ear inhibited 12-O-tetradecanoylphorbol-13-acetate (1 micrograms/ear)-induced mouse ear edema in a dose-dependent manner with a 50% effective dose of 28 micrograms/ear (13-63 micrograms/ear, n = 10/dose), but did not improve arachidonic acid (4 mg/ear)-induced mouse ear edema at 1 mg/ear. These results suggest that YM-26567-1 is a competitive PLA2 inhibitor showing a higher affinity for group II than group I PLA2, and that it may act as a potent anti-inflammatory compound through its direct inhibition of PLA2.

Hexokinase II prepared from Ehrlich-Lettre hyperdiploid tumor cells (ELD cells) was subjected to a limited digestion by trypsin. After 60 min digestion, hexokinase II (100 kDa) was completely cleaved to two fragments with the molecular weight of about 60 kDa and 40 kDa as manifested in SDS-PAGE. It was noteworthy that the enzyme activity was observed even at the time when the native enzyme molecule was no more detectable. These fragments were separated by SDS-PAGE irrespective of the presence of a reducing agent, but neither by native PAGE nor by cellulose acetate membrane electrophoresis under the nondenaturing conditions. Neither kinetic parameters such as Km values for ATP and glucose nor an ability of binding to mitochondria were changed significantly by the tryptic digestion. These results indicate that an essential conformation of hexokinase II can be restored by the self-association of two fragments produced as a result of the cleavage by trypsin at the middle of the molecule. Affinity labeling with 2',3'-dialdehyde ATP followed by the trypsin digestion showed that ATP binding site resided in the 40 kDa fragment. Furthermore, the mode of the response in the incorporation of this ATP analog to hexose phosphate, moreover, was similar to that in the catalytic activity.

The healing process of bone fractures produced in rat mandibulae was examined with conventional X-ray radiography and xeroradiography. Sixty rats of about 200g were offered to this examination and fractures were experimentally produced in the right mandibulae. Radiological observations were carried out immediately, 1 day, 4 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days after fracturing. The conventional X-ray radiograms showed no noteworthy changes until the 4th day after fracturing. The radiopacity gradually increased from the 7th Day until the 56th day. The xeroradiograms obtained on the 4th day showed some early changes which appeared on the periosteal regions of the right mandibulae. The calcification of fractured regions on the xeroradiograms gradually increased until the 56th day, similar to that observed on the conventional X ray radiograms.