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Alkaline extract of Sasa senanensis Rehder (SE) has shown diverse biological activity. As an extension, whether SE affects the function of activated macrophages was investigated. SE inhibited the nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells. Western blot and RT-PCR analyses demonstrated that this was due to the inhibition of inducible NO synthase (iNOS) expression at both protein and mRNA levels. ESR spectroscopy shows that SE dose-dependently scavenged the NO radical produced by NOC-7. In order to confirm the anti-inflammatory potency, possible effects on prostaglandin (PG) E(2) production and expression of enzymes involved in the arachidonic acid pathway were next investigated. It was found that SE effectively inhibited the PGE(2) production by LPS-stimulated RAW264.7 cells, although the extent of inhibition of PGE(2) was slightly less than that of NO production. SE inhibited cyclooxygenase (COX)-2 expression at both protein and mRNA levels, but to much lesser extents as compared with those for iNOS expression. SE contained much lower concentration of arginine, precursor of NO, as compared with the culture medium. These data suggest that SE exerts a weak anti-inflammatory activity.

The tumor-specific cytotoxicity and the type of cell death induced by thirty-eight newly synthesized tetrahydroisoquinoline derivatives in human oral squamous cell carcinoma cell lines were investigated. 6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)(3,4-dimethoxyphenyl) methanone that has bulky substituents (such as 3,4-dimethoxybenzoyl group) (TQ9) and ethyl 2-benzyloxycarbonyl-1,2,3,4-tetrahydroisoquinoline-1-carboxylate that has ethoxycarbonyl group and benzyloxycarbonyl group (TD13) showed the highest tumor-specific cytotoxicity (TS=12.5 and 5.3, respectively). This supports the importance of molecular size for the cytotoxicity induction. TQ9 induced internulceosomal DNA fragmentation and caspase-3 activation only marginally in HL-60 cells, whereas it enhanced the formation of acidic organelles (stained with acridine orange) without induction of DNA fragmentation or caspase-3 activation in human squamous cell carcinoma cell lines (HSC-2, HSC-4), suggesting the induction of autophagy in the latter cells.

We previously reported that Sairei-to concentration-dependently modified lipopolysaccharide (LPS)-stimulated prostaglandin E(2) (PGE(2)) production in mouse macrophage-like RAW264.7 cells. Among twelve major ingredients of Sairei-to, Scutellariae radix inhibited the LPS-stimulated PGE(2) production to the greatest extent, followed by Zingiberis rhizoma, Glycyrrhizae radix, Atractylodis lanceae rhizoma and Pinelliae tuber. Scutellariae radix contained several major flavonoids such as baicalin, baicalein and wogonin. We investigated the effect of these flavonoids on PGE(2) production and COX-2 expression by LPS-activated RAW264.7 cells. Wogonin inhibited PGE(2) production most efficiently, followed by baicalein and then baicalin, in the same order as their membrane permeability. It was unexpected that wogonin and all other compounds would fail to inhibit the expression of COX-2 at both protein and mRNA levels, suggesting the importance of re-evaluating the point of action of wogonin.

Three Chinese herbal extracts, Drynaria baronii, Angelica sinensis and Cornus officinalis Sieb. et Zucc (referred to as DB, AS, CO, respectively), were investigated for their possible anti-inflammatory activity. DB, AS and CO inhibited nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like RAW264.7 cells. Western blot and RT-PCR analyses demonstrated that this was due to the inhibition of inducible NO synthase (iNOS) expression at both protein and mRNA levels. Electron-spin resonance spectroscopy showed that DB, AS and CO dose-dependently scavenged the NO radical produced by NOC-7 in the presence of carboxy-PTIO. In order to confirm the anti-inflammatory potency, effects on prostaglandin (PG) E(2) production and the expression of enzymes involved in the arachidonic acid pathway were next investigated. DB and CO effectively inhibited the PGE(2) production by LPS-stimulated RAW264.7 cells, although the extent of inhibition of PGE(2) production was slightly lower than that of NO production. AS only marginally inhibited the LPS-stimulated PGE(2) production. DB, AS and CO inhibited cyclooxygenase (COX)-2 expression at both protein and mRNA levels, but to much lesser extents as compared with that for iNOS expression. These data further substantiate the anti-inflammatory potency of DB, AS and CO.

Mastic is a resinous exudate obtained from the stem and the main leaves of Pistacia lentiscus. We have reported the antiplaque effect of mastic-containing chewing gum on the oral cavity. We hypothesize that mastic may be a multifunctional food which has some beneficial pharmaceutical properties. The aim of this study was to assess the biological activity of solid and liquid types of mastic by cytotoxicity against fibroblasts, radical-scavenging activities and inhibitory effect on cell death of oral polymorphonuclear leukocytes (OPMNs). Mastic showed selective antibacterial action against Porphyromonas gingivalis and Prevotella melaninogenica, but no anti-HIV activity. Among a total of thirteen human cell types, promyelocytic leukemia HL-60 was the most sensitive to the cytotoxicity of mastic, followed by myeloblastic leukemia (ML-1, KG-1), erythroleukemia (K-562), oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), hepatocellular carcinoma (HepG2), glioblastoma (T98G, U87MG) and normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast, most resistant). Mastic did not induce the differentiation of myelogenous leukemic cells into maturing cells with higher nitroblue tetrazolium-reducing activity, but induced apoptotic cell death, characterized by internucleosomal DNA fragmentation, caspase-3 activation and a decline in the intracellular concentration of putrescine. The cytotoxicity of mastic against leukemic cells did not diminish during its storage. On the other hand, mastic inhibited the spontaneous apoptosis of OPMNs. Mastic showed hydroxyl radical-scavenging activity. The selective antibacterial and apoptosis-modulating activity of mastic suggests its possible beneficial effects on oral health.

A total of twenty benzo[b]cyclohept[e] [1,4]oxazines and their S-analogs, and 2-aminotropone derivatives were investigated for their cytotoxicity against three human normal cells and four tumor cell lines. These compounds showed moderate tumor-specific cytotoxicity. The cytotoxicity was enhanced by bromination at the tropone ring and replacement by formylbenzene. The cytotoxicity of 2-(2-hydroxyanilino) tropone was enhanced by introduction of bromine or isopropyl group to the tropone ring. The presence of a hydroxyl group at ortho or para-position should be necessary for the appearance of cytotoxicity and tumor-specificity. The highly active derivatives, 7-bromo-2-(4-hydroxyanilino)tropone [16] and 4-isopropyl-2-(2-hydroxyanilino)tropone [20], induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in human promyelocytic leukemia HL-60 cells, but only at concentrations twice or four times higher than CC(50) values. These compounds induced no discernible DNA fragmentation, and activated caspases much more weakly in human oral squamous cell carcinoma HSC-2 cells. Both [16] and [20] failed to induce the production of acidic organelles, a marker of autophagy, in contrast to the nutritional starvation. These data demonstrated that 2-aminotropones showed relatively higher tumor-specificity than benzo[b]cyclohept[e] [1,4]oxazine, and that 2-aminotropones induced little or no apoptotic cell death in oral squamous cell carcinoma, in contrast to HL-60 cells.

A total of thirty-nine naphtho[2,3-b]furan-4,9-diones and related compounds were tested for their cytotoxicity against three human normal oral cells (gingival fibroblast, HGF, pulp cell, HPC, periodontal ligament fibroblast, HPLF) and four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). 2-Acetylnaphtho[2,3-b]furan-4,9-dione [1] was highly cytotoxic to both normal and tumor cells, yielding low tumor-specificity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan [4], the 2-(3-furanoyl) benzoic acids [5, 6] and the 1,4-naphthoquinones [7, 8] showed much reduced cytototoxicity and low tumor-specificity. The introduction of phenoxy [18], isopropylamino [23] or 2-methylpiperidino [33] groups to the 2-position of naphtho[2,3-b]furan-4,9-dione yielded compounds that showed the greatest tumor-specificity. These compounds, at twice or four times higher concentrations than CC50, induced the activation of caspase-3, caspase-8 and caspase-9 in the HSC-2 and HL-60 cells, but not so apparently in the HSC-4 cells. However, they did not induce internucleosomal DNA fragmentation in the HSC-2 and HSC-4 cells even after 24 hours incubation and only slightly induced DNA fragmentation in the HL-60 cells. Compound [18] induced the production of annexin-positive cells, but did not induce microtubule-associated protein light chain 3 (LC3) accumulation in autophagosomes in LC3-green fluorescent protein (GFP)-transfected HSC-2 cells. These data suggested that naphtho[2,3-b]furan-4,9-diones may induce the early apoptotic marker, without induction of caspase activation and DNA fragmentation in oral squamous cell carcinoma cell lines. Quantitative structure-activity relationship (QSAR) analysis suggests the applicability of the theoretical calculations such as frontier molecular orbital, dipole moments and hydrophobicity in predicting their cytotoxic activity.

We previously reported that alpha-trifluoromethyl acyloins (TFs) induced various types of cell death, depending on the target cancer cell line. We investigated here what type of cell death is induced by a-trifluoromethyl acyloins in two human oral squamous cell carcinoma cell lines (HSC-2, HSC-4). TFs produced few TUNEL-positive cells. TFs induced annexin V/PI-double positive HSC-2 cells and annexin V-positive/PI-negative HSC-4 cells, respectively, but failed to activate caspase-3, capase-8 and caspase-9 in both HSC-2 and HSC-4 cells. On the other hand, TFs induced the formation of acidic organelles (detected by acridine orange staining) in both HSC-2 and HSC-4 cells. When HSC-2 and HSC-4 cells that had been transfected with expression vector encording the microtubule-associated protein 1 light chain 3 (LC3) gene fused to green fluorescent protein (GFP) were treated with TFs, LC3-GFP fusion protein was accumulated as granular dots in autophagosomes. Pretreatment with 3-methyladenine, an inhibitor of autophagy, partially inhibited the cytotoxicity of TFs, the formation of acidic organelles and LC3 accumulation in the autophagosome. These data suggest that alpha-trifluoromethyl acyloins may induce autophagic cell death in HSC-2 and HSC-4 cells following the early stage of necrosis or apoptosis, respectively.

BACKGROUND Direct measurement of plasma nitric oxide (NO) concentration is possible with a newly developed catheter-type sensor. METHODS AND RESULTS Adult mongrel dogs (n=5) were fed a high-fat diet (120 kcal . kg(-1) . day(-1)) for 8 months, then endothelial function was assessed by the change in NO concentration induced by acetylcholine (ACh) (DeltaNO). Simultaneously, average peak velocity (APV) was obtained by Doppler guide wire. Although fasting plasma glucose levels did not change after high-fat diet, fasting plasma insulin levels increased significantly (103+/-36 vs 106+/-27 mg/dl, P=0.89 and 0.15+/-0.15 vs 0.26+/-0.07 ng/ml, P=0.04, respectively). ACh-induced peak APV after high-fat feeding was not significantly different from that at baseline (ACh 0.1 microg/kg; 43+/-17 vs 51+/-7 cm/s, P=NS, ACh 0.4 microg/kg; 45+/-20 vs 47+/-16 cm/s, P=NS, respectively). The DeltaNO was significantly smaller after high-fat diet than at baseline (ACh 0.1 microg/kg; 2.6+/-1.6 vs 1.0+/-0.5 nmol/L, P=0.03, ACh 0.4 microg/kg; 3.8+/-2.3 vs 1.8+/-1.1 nmol/L, P=0.04, respectively). CONCLUSIONS In high-fat-diet-induced obese dogs NO production was impaired in the early stage when the coronary flow response to ACh may be preserved.

Tetradecane (TD), testosterone (TS), and dimethyl sulfoxide (DMSO) were separately inuncted on rabbit pinnas once a day; the pinnas were biopsied on days 1, 3, 7, and 28. Untreated pinnas and squalane-treated pinnas were used as controls. Three-dimensional images of sebaceous glands were reconstructed from their serial histologic sections using a computer-image analysis system. By stereometry, the sebaceous gland volumes gradually increased during the course of treatments with TS and DMSO, whereas the volumes of TD-treated sebaceous glands increased, reached the maximum on day 7, and then decreased. The acinus number was increased by all the substances, the DMSO-treated sebaceous glands on day 28 showing the largest number of acini. The volumes of individual acini increased until day 28 with TS treatment, whereas with TD and DMSO treatments they increased, reached the maximum on day 7, and then decreased. Stereographically, each untreated control sebaceous gland was composed of 20-30 pear-shaped acini. On day 28, acini of TS-treated sebaceous glands were swollen and ovoid in shape, DMSO-treated sebaceous glands resembled a bunch of grapes, and acini of TD-treated sebaceous glands were widely scattered and showed globular swelling arising from an elongated tube. Hyperplastic responses of sebaceous glands are quite different according to substances applied topically.

Thirty-two cases of gastric cancer associated with hepatic cirrhosis were reviewed, retrospectively. These patients represented 1.3% of 2590 patients with gastric cancer who were treated over the 23 year period from 1966 to 1988. The male to female ratio was 9.7:1 for gastric cancer patients with hepatic cirrhosis (mean age, 63.2 years) and 2:1 for all patients with gastric cancer (mean age, 61.2 years). Among the 32 patients, one was inoperable, and 31 patients underwent surgical exploration. The gastric cancers were resected in 28 patients (90.3%), while the cancers in 3 patients (9.7%) were non-resectable. Twenty-two patients underwent curative resections with a five-year survival rate of 51%. Postoperative complications occurred in 6 patients (20%), but no anastomotic leakage occurred. There were no operative mortalities. On the basis of the present study, different surgical procedures are indicated depending on the preoperative hepatic conditions for patients with gastric cancer accompanied by hepatic cirrhosis.

Experimental studies have revealed that duodenogastric reflux subsequent to gastrectomy was one of the most significant factors for the development of carcinoma in the remnant stomach. For this reason, the binding of carcinogenic agents to the nucleic acids in the gastric mucosa exposed to the duodenal contents were studied using an organ culture method. The intracellular DNA levels bound with carcinogenic agents were significantly increased in the mucosa of the remnant stomach, as compared to the parietal mucosa in the whole stomach. The remnant stomach may therefore provide an environment that enhances the development of cancer.

From 1978 to 1989, a total of 1272 patients with gastric cancer were admitted to Kurume University Hospital. Of these, 1184 (93%) underwent gastric resections. Of the resected cancers, 140 cases were gastric cancers invading the esophagus, as confirmed by postoperative pathological studies. For all 168 gastric cancers invading the esophagus, the resectability rate was 83% (140/168). These cancers were subdivided chronologically, with those during 1978-1983 included in Group 1 and those during 1984-1989 in Group 2. Group 2 patients were treated with a higher proportion of aggressive extended radical operations, such as combined resections of the diaphragm and lymph node dissections as far as the paraaortic lymph nodes (N4). The resectability rate was 76% in Group 1 and 93% in Group 2, due to the more aggressive operations. A lower esophagectomy with a total gastrectomy, mainly from a left thoracoabdominal approach, was performed for 71% of the patients in Group 1, and for 90% in Group 2. Lymph node metastasis was related to the extent of esophageal invasion and inversely related to the 5 year-survival rate. In all cases of esophgeal invasion of less than 1 cm, there were no mediastinal lymph node metastases, and the 5 year-survival rate was 76%. On the other hand, when esophageal invasion was more than 4cm, 50% had mediastinal lymph node metastases and the 5 year-survival rate was zero. The tumor size was also related to lymph node metastasis. When the maximal tumor length was less than 5cm, a negative lymph node metastasis was found in 78% of cases, and the 5 year-survival rate was 76%. On the other hand, when the tumor length was more than 10 cm, only 6% had a negative lymph node metastasis and the 5 year-survival rate was only 15%. The 5 year-survival rate with curative resection was 45% for Group 1, and 71% for Group 2. For those at stage III, 12% of Group 1 could be curatively resected with an extended radical operation, 46% of Group 2 (p

Seventy-six lesions of 38 cases of multiple early gastric cancer were clinicopathologically studied, and the localization of CEA and CA19-9 in the lesions was also investigated using enzyme antibody method. Multiple early gastric cancer (double cancer) accounted for 5.5% of all resected gastric cancers, and was predominant in elderly men, and in region A and M. Macroscopically, the major lesions predominantly compresied torus type, while the minor lesions, concave type. Histologically well differentiated tubular adenoma was predominant in both lesions. Twelve of 38 (31.6%) minor lesions occupied oral-side regions from the major lesions, and 11 minor lesions were comparised of minimal size of cancers. The importance of appropriate decision of the resection area should be emphasized not to leave the minor lesions in the residual stomach. As for tumor markers, both CEA and CA19-9 were positive in all lesions of poorly differentiated cancers. On the other hand, CEA positive rates in well defferentiated cancers were 80.6% in the major and 56.3% in the minor lesions, and CA19-9 positive rates, 64.5% and 50.0% respectively. Both tumor markers showed higher positive rates in major lesions rather than those in the others, which might reflect a fact that, with the lapse of time, the cancers gradually proliferated, differentiated, and produced the tumor markers.

Since 1985, we have been performing an extensive combined resection of the unilateral diaphragm under left thoracotomy in cases of gastric cancer with esophageal invasion, involving invasion to the serosa without distant or disseminated metastasis. From 1976 to 1988, the resection was performed in 111 cases of upper gastric cancer with esophageal invasion. When classified by the approach route, there were 77 cases of left thoracoabdominal approach and mediastinal incision, and 34 of abdominal approach alone. Of the 77 cases of left thoracoabdominal approach, combined resection of the diaphragm was performed in 22, and the surgical results were evaluated form 17, after excluding 3 of only partial resection of the diaphragm, one of remnant gastric cancer and one of multiple cancer. The 2 year survival rates all ps (+) cases were 64% for combined resection and 41.8% for non-combined resection. When classified by stage, the survival rates in stage IV cases were 55.6% for combined resection, and 12.5% for non-combined resection, showing a significant different.

During the 14 years from 1976 to 1989, 1442 gastric cancers were resected in the Department of Surgery at Kurume University. 546 (37.9%) were primary early gastric cancers. One (0.2%) of these was associated with a synchronous liver metastasis. In the advanced gastric cancers, the rate of synchronous liver metastasis was 12.8%. However, synchronous liver metastasis with an early gastric cancer is quite rate. Only 19 cases have been reported in the Japanese literature. A case involving an early cancer, 10 mm in diameter, that was associated with liver metastasis is reported.

We experienced a case of early gastric carcinoma of type IIc occurred 36 years after gastrojejunostomy in the pyloric stenosis due to gastric ulcer.This case is a 56-year-old man. A concave lesion and concentrated pattern of mucous membrane connecting to the gastrojejunostomy regions of the greater curvature of corpus ventriculi were noted. The patient was diagnosed as having a signet-ring cell carcinoma by endoscopic biopsy. Gastrectomy of the pyloric side including anastomotic regions was carried out, and reconstructed by Roux-en-Y method. The macroscopic type of the extirpated sample was early gastric cancinoma of type IIc and histologically it was in H0P0n0 m: stage I. The tissue type was signet-ring cell carcinoma. With regard to the developmental mechanisms of gastric carcinoma after gastrojejunostomy, a chemical factor due to stimulus of duodenal juice is the most important. Endoscopy is useful for the diagnosis of the disease, especially, changes of surroundings of the anastomotic region should be marked in the follow up of stomach performed gastrojejunostomy.

Two rare cases of a spontaneous portacaval shunt occurring in patients with gastric cancer and hepatic cirrhosis are retrospectively reviewed with special attention for the operative indications and techniques. One patient underwent proximal gastrectomy for a IIa cancer in the C region with splenectomy and closure of the splenorenal shunt, and died two years and 3 months later due to hepatic failure. The other patient underwent distal gastrectomy for IIa + IIc, IIc double cancers in the M and A regions with splenectomy through an upper transverse abdominal incision that ligated and divided the paraumbilico-caval shunt, and is alive and well 1 year and 2 months, postoperatively. The ICG-R15 was markedly decreased and the K-ICG and ICG-Rmax were improved following the operation in both cases. The preoperative chronic encephalopathy and hyperammonemia disappeared postoperatively in both cases.

During the 12 years from 1977 to 1988, a total of 425 cases of early gastric cancer (EGC) were treated by resection. Of these, 53 (13%) had lymph node metastases, occurring in 6 cases of mucosal cancer (m-cancer) and 47 cases of submucosal cancer (sm-cancer). Among the patients with positive lymph node metastases, 46 (87%) had metastases in the Group 1 perigastric regional nodes, and 8 (2%) had metastases in Group 2 lymph node. These data support the general view that lymph node metastasis occurs in only 2-5% of m-cancers and 15-20% of sm-cancers, and then mainly in Group 1 lymph nodes. A rare case of early cancer with Group 3 juxta regional lymph node metastasis (n3+) is described in this report with special attention to the surgical management and postoperative chemotherapy.

Twenty-six benzocycloheptoxazine derivatives were investigated for their effect on nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Benzo[b]cyclohepta[e][1,4]thiazine most effectively inhibited the LPS-stimulated NO production at noncytotoxic concentrations. 6H-Benzo[b]cyclohepta[e][1,4]-diazine cation, and benzo[b]cyclohepta[e][1,4]oxazine and its 6-bromo derivative also efficiently inhibited the LPS-stimulated NO production. Another sixteen benzo[b]cyclohepta[e]-[1,4]oxazine derivatives, 14H-[1,4]benzoxazino[3',2' :3,4]-cyclohepta[1,2-b][1,4]benzoxazine and its 7-bromo- and 7-isopropyl derivatives were slightly less active (selectivity index (SI)=83-66). Bromination of benzo[b]cyclohepta[e][1,4]-thiazine, benzo[b]cyclohepta[e][1,4]oxazine and 2-methylbenzo[b]cyclohepta[e][1,4]oxazine at C-6, C-8 or C-10 positions resulted in the significant reduction of the inhibitory activity. The observed inhibitory activity of benzo[b]cyclohepta-[e][1,4]thiazine and its 6,8-dibromo derivatives were not due to the reduction of the intracellular level of inducible NO synthase protein (based on Western blot analysis), nor to NO scavenging activity (based on ESR spectroscopy). These results suggest the possible anti-inflammatory action of benzocyclo-heptoxazines via inhibition of LPS-activated macrophages.

The cytotoxic activity of sodium 5,6-benzylidene-L-ascorbate (SBA) against eight human cancer cell lines and three human normal cells was investigated, SBA showed slightly higher cytotoxicity against human tumor cell lines, as compared with normal cells, with a tumor-specificity index of 2.0. The human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to SBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human oral normal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) were the most resistant. In contrast to actinomycin D, SBA induced little or no activation of caspase-3, caspase-8 and caspase-9 in the HSC-2, HSC-4, T98G and HL-60 cells, regardless of incubation time (either 6 or 24 h). SBA induced little or no internucleosomal DNA fragmentation after 6 h in all of these cells. However, prolonged treatment with SBA (24 h) induced a smear pattern of DNA fragmentation in the HSC-2, HSC-4 and T98G cells and a low level of internucleosomal DNA fragmentation in the HL-60 cells. Electron microscopy demonstrated the destruction of mitochondrial structure and autophagocytosis of broken organelles by SBA in the HSC-2, HSC-4 and HL-60 cells. At higher concentrations of SBA, necrotic cell death was observed in the HSC-2 cells, but not in the T98G cells, where the production of acidic organelles (detected by acridine orange staining) was much lower than that attained by nutritional starvation, a well-defined method of inducing autophagy. The present study suggests that SBA induces various degrees of autophagic cell death, followed by either necrosis or apoptosis at laters stage, depending on the cell type.

Glycyrrhizin is a major constituent of Kanzo, a popular herbal medicine used in food and cosmetics. Glycyrrhizin alone did not stimulate nitric oxide (NO) or prostaglandin E2 (PGE2) production by RAW 264.7 cells, but modified lipopolysaccharide (LPS)-stimulated NO and PGE2 production in a bimodal fashion: it was stimulatory at lower concentrations, whereas it was inhibitory at higher concentrations. Electron-spin resonance spectroscopy showed that glycyrrhizin slightly scavenged the superoxide anion generated by hypoxanthine-xanthine oxidase reaction, but did not scavenge the DPPH and NO radicals. The present study demonstrates the concentration-dependent action of glycyrrhizin on both PGE2 and NO production by activated macrophages.

Sairei-to and its twelve ingredients were investigated for their activity to stimulate prostaglandin E2 (PGE2) production by unstimulated and lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. LPS significantly stimulated the production and extracellular secretion of PGE2 by RAW 264.7 cells. Sairei-to concentration-dependently modified the LPS-stimulated PGE2 production. Among Sairei-to ingredients, Scutellariae radix inhibited the LPS-stimulated PGE2 production to the greatest extent, followed by Zingiberis rhizoma, Glycyrrhizae radix, Atractylodis lanceae rhizoma and Pinelliae tuber. On the other hand, Bulpeuri radix, Alismatis rhizoma, Zizyphi fructus, Polyporus, Hoelen, ginseng radix and Cinnamomi cortex further enhanced the LPS-stimulated PGE2 production. Western blot analysis demonstrated that Sairei-to unexpectedly enhanced the expression of cyclooxygenase-2 (COX-2) protein level, but did not significantly affect phospholipase A2 protein level. The present study suggests that the modification of the enzyme activity of COX-2 may be involved in the concentration-dependent effect of Sairei-to on PGE2 production by macrophages.

We have previously reported that lignin carbohydrate complex and E. coli lipopolysaccharide (LPS), but not their precursors or (-)-epigallocatechin-3-gallate (EGCG), enhanced the nitric oxide (NO), citrulline and asparagines production by the mouse-macrophage-like cell line Raw264.7. Here the EGCG inhibition of NO production by LPS-stimulated Raw264.7 cells is reported. EGCG induced apoptotic cell death characterized by internucleosomal DNA fragmentation, whereas it inhibited the formation of secondary lysosomes and autophagosomes detected as granular dot-like distribution of acridine orange and microtubule-associated protein light chain 3 fused with green fluorescent protein (LC3-GFP). Autophagy inhibitors such as 3-methyladenine and bafilomycin A1 induced apoptotic cell death in the Raw264.7 cells. The inhibition of macrophage NO production by EGCG may be due to the apoptosis induction coupled with autophagy inhibition.

We have previously reported that Rikko-san (RKS) inhibited the lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 in mouse macrophage-like RAW264.7 cells without affecting the expression of cyclooxygenase (COX)-2. Here RKS inhibition of the enzyme activity of both COX-1 and COX-2 proteins was investigated. Western blot analysis showed that RKS did not significantly change the S-nitrosylated COX-2 protein level. On the other hand, RKS inhibited the PG production catalyzed by purified COX-2, more effectively than that catalyzed by purified COX-1. These results suggest that RKS inhibits the PGE2 production by selectively inhibiting the COX-2 activity in activated macrophages.

Twenty-six benzocycloheptoxazine derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). Benzo[b]cyclohepta[e][1,4]thiazine [1] exhibited very weak cytotoxicity, whereas its 6,8,10-tribromo derivative [3] exhibited higher cytotoxicity and tumor specificity (TS = 5.6). 6H-Benzo[b]cyclohepta[e][1,4]diazine [4] and its cation [5] exhibited no tumor specificity. Among eighteen benzo[b]cyclohepta[e][1,4]oxazine derivatives [6-23], 6,8,10-triboromo- [9], 6-bromo-2-methyl- [20], and 6-bromo-2-chloro- [21] derivatives showed the highest tumor-specific cytotoxicity (TS = 12.5, 9.1 and 11.5, respectively). 14H-[1,4]Benzoxazino[3',2':3,4]cyclohepta[1, 2-b][1,4]benzoxazine [24] and its 7-bromo- [25] and 7-isopropyl- [26] derivatives had much lower cytotoxicity and tumor-specificity. Compounds [9, 20, 21] at 50% cytotoxic concentration (CC50) induced internucleosomal DNA fragmentation and caspase activation in HL-60 cells. On the other hand, these compounds induced apoptosis only at concentrations higher than CC50 in HSC-2 cells and failed to induce apoptosis in HSC-4 cells. Compounds [9, 20, 21] induced the formation of acidic organelles as measured by acridine orange staining. Transmission electron microscopy demonstrated the induction of moderate enlargement of mitochondria, the endoplasmic reticulum and nuclear membrane, and the vacuolation of the endoplasmic reticulum and the presence of a number of lamellar body-like organelles. These results indicate the diversity of the type of cell death induced by benzocycloheptoxazine derivatives in human tumor cell lines.

Fourteen vitamin K2 (menaquinone (MK)-n, n = 1-14) and ten prenylalcohol derivatives (n = 1-10) with different numbers (n) of isoprenyl groups in the side chains were investigated for their cytotoxicity against nine human tumor cell lines and three human normal oral cells. Among the vitamin K2 derivatives, MK-2 (n = 2) showed the greatest cytotoxicity, followed by MK-1 (n = 1) and MK-3 (n = 3). MK-1, MK-2 and MK-3 showed the highest tumor-specific index (TS=2.0, 2.0 and1.7, respectively). Among the prenylalcohols, geranylgeraniol (GG) (n = 4) showed the highest cytotoxicity, followed by farnesol (n = 3) and geranylfarnesol (GF) (n = 3). GG showed the highest tumor-specificity (TS = 1.8), followed by farnesol (TS =1.4), GF (TS=1.3). However, the tumor-specificity of MK-2 and GG was much lower than that of conventional chemotherapeutic agents. The human leukemic cell lines were the most sensitive, whereas the human glioblastoma cell lines were the most resistant to MK-2 and GG. MK-2 did not induce internucleosomal DNA fragmentation in either the human promyelocytic leukemia HL-60 or the human squamous cell carcinoma HSC-4 cell lines. GG induced marginal internucleosomal DNA fragmentation in the HL-60 cells, but not in the HSC-4 cells. Both MK-2 and GG did not induce the formation of autophagosomes, nor did they clearly change the intracellular concentration of three polyamines. Electron spin resonance (ESR) spectroscopy showed that only MK-1 (n = 1), as well as GGF (n = 7) and GFF (n = 8) which had lower cytotoxicity, produced radicals, suggesting the lack of connection between cytotoxicity and radical production. The present study demonstrates that the presence of 1,4-naphtoquinone structure (including alpha,beta-unsaturated ketones) in vitamin K2 derivatives confers on them the ability to induce non-apoptotic cell death.

The effect of 20 trihaloacetylazulene derivatives with one halogen atom, on nitric oxide (NO) production by mouse macrophage-like cells Raw 264.7 was investigated. 2-Methoxyazulenes and 2-ethoxyazulenes exhibited comparable cytotoxicity. Trichloroacetylazulenes generally exhibited higher cytotoxicity, as compared with the corresponding trifluoroacetylazulenes. Substitution of chloride, bromide or iodine at the C-3 position further enhanced their cytotoxicity. All of these compounds failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but did inhibit NO production by LPS-activated Raw 264.7 cells to different extents. 1-Trichloroacetyl-2-methoxyazulene and 1-trichloroacetyl-2-ethoxyazulene, with less cytotoxic activity, inhibited NO production to the greatest extent, producing the highest selectivity index (SI) of24.7 and28.7, respectively. This was accompanied by the efficient inhibition of inducible NO synthase (iNOS) mRNA expression, but not by iNOS protein abundance. Electron spin resonance (ESR) spectroscopy showed that neither of these compounds produced radicals, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study suggests that the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages might be derived from the perturbation of NO anabolism (inhibition of iNOS mRNA expression and possibly the inactivation of iNOS protein) rather than NO catabolism (NO scavenging).

The cytotoxicity of beta-cyclodextrin benzaldehyde inclusion compound (CDBA) against human normal and cancer cell lines was investigated. CDBA showed slightly higher cytotoxicity against human tumor cell lines, as compared to normal cells, with a tumor-specificity index of 2.2. Human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to CDBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human normal cells (gingival fibroblasts, pulp cells, periodontal ligament fibroblasts) were the most resistant. CDBA induced internucleosomal DNA fragmentation in HL-60 cells and caspase-3, -8, -9 activation, but to a much lesser extent than that attained by UV irradiation or actinomycin D. On the other hand, CDBA did not induce DNA fragmentation, nor caspase activation in HSC-2, HSC-4 or T98G cells. Electron microscopy demonstrated that CDBA induced the destruction of mitochondrial structure and digestion of broken organelles by secondary lysosomes in all of these cells. CDBA also increased the number of acidic organelles as judged by acridine orange staining. The present study suggests that CDBA induces autophagic cell death in cancer cell lines.

Phenoxazines have shown diverse biological activities, but tumor-specific cytotoxic activity has not been investigated. A total of 24 phenoxazine derivatives (WM1-24) was investigated for their relative cytotoxicity against human tumor cell lines vs. normal cells. WM7 and WM8 showed the highest tumor-specificity index of 4.3 and 4.8, respectively. Considerable difference in drug-sensitivity was found among these tumor cell lines. Human promyelocytic leukemia HL-60 cells showed the highest sensitivity to both WM7 and WM8, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), and human gingival fibroblast (HGF), pulp cell (HPC) and periodontal ligament fibroblast (HPLF) were the most resistant. WM7 and WM8 induced little or no internucleosomal DNA fragmentation, and activated caspase-3 in HSC-2, HSC-4 and human glioblastoma T98G cells. These compounds failed to induce autophagic cell death, as judged by acridine orange and microtubule-associated protein 1 light chain 3 (LC3)-GFP assays. These results suggested that the higher cytotoxicity of WM7 and WM8 are derived from the positively-charged quaternary nitrogen substituents on the phenoxazine ring and the electron density of nitrogen at N12, and that inhibition of autophagy is not always coupled with apoptosis induction.

Fourteen 4-trifluoromethylimidazole derivatives were investigated for their cytotoxicity against three human normal cells (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF) and four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4 and promyelocytic leukemia HL-60). Among these compounds, 4-trifluoromethyl-1,2-diphenylimidazole (IM5), 1-benzyl-4-trifluoromethyl-2-phenylimidazole (IM7) and 5-[1-ethoxy-2,2,2-trifluoro-1-(trifluoromethyl) ethyl]-1-methyl-2-phenyl-1H-imidazole (IM12) showed much higher cytotoxicity and tumor-specificity than the other compounds. IM5, the most potent compound, induced different types of cell death depending on the target cells. IM5 induced DNA fragmentation of oligonucleosomal units (a biochemical hallmark of apoptosis) in the HL-60 cells, but not in such a clear-cut laddering pattern in the HSC-2 cells. On the other hand, IM5 produced secondary lysosomes digesting broken organelles, without induction of internucleosomal DNA fragmentation and disappearance of cell surface microvilli in the HSC-4 cells, even though the HSC-2 and HSC-4 cells showed comparable sensitivity to IM5. These data suggest that the type of cell death is determined by the type of target cells, but not by the drug-sensitivity of the cells.

The plucked hairs and biopsied hair follicles of the scalp were obtained from a female patient with monilethrix. By scanning electron microscopy, the plucked hairs showed a typical moniliform feature composed of alternated nodes and inter- nodes. By computer stereography, reconstructed three-dimensional models of in vivo hair structures showed that the diameter of hair shaft was partially reduced in the keratogenous zone and that the reduction was severe in the hair cortex and cuticle layers but mild in the inner root sheath. By transmission electron microscopy, a significant degeneration of hair matrix cells was found, and a zig-zag disarray of cortical tonofibrils and invaginations of the hair cuticle cells into the cortex were noticed in the suprabulbar portion. In some hairs, the hair bulbs showed no degeneration, but a degeneration of cortical cells and invaginations of hair cuticle were seen in some portions in the developing zone of the cortex. This suggests that not every hair matrix is damaged in a synchronized fashion, but individual hairs are affected independently. Furthermore, in all the hair and hair follicles examined, cytoplasmic vacuolations were always seen in the various layers, and abnormal formation of tonofibrils was often observed in the cortex. In monilethrix, a cell abnormality may inherently be present in the hair tissue and, when such abnormality occurs severely, the cortical cells are particularly affected in the hair matrix. This seems to result in. a decrease in number of cortical cells and thinning of the hair shaft.

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.

From 1979 to 1988, a total of 1265 cases of gastric cancer were admitted to our Institute. Of these, 80 cases (6.3%) involved hepatic metastasis. Thirty-eight cases (47.3%) underwent gastrectomy (24 distal, 2 proximal, 11 total and one partial). Of these 38 cases, 7 underwent a combined resection of a simultaneous metastatic hepatic tumor. A total of 19 (67.9%) of the 28 cases in H1, 6 (37.5 %) of the 16 cases in H2, and 13 (36.1%) of the 36 cases in H3 underwent gastrectomy. Hepatic metastasis occurred most frequently (18.2%) in Borrmann type 1, 5 % in type 2, 14.9% in type 3 and 8.7% in type 4. Of all 593 resected advanced cases, there were 283 differentiated type, with a hepatic metastasis rate of 14.5%, and 310 undifferentiated type with 7.1 % rate of hepatic metastasis. In H1 and H2, the prognosis after primary gastric cancer resection was better than for unresected cases. Moreover, there have been reports recently of long-term survival using continuous hepatic arterial infusion of anti-cancer durgs, and of combined resection of the metastatic hepatic tumor after curative gastrectomy. Therefore we should not give up these H1 and H2 patients.

We report 9 rare cases (7 males and 2 females) of early gastric cancer of the esophagogastric junction. From 1976 to 1988, 1308 cases of gastric cancer were resected in our Institute. Of these, 479 (36.6%) cases were early gastric cancer. Among all 479 early gastric cancers, 9 (1.9%) were located within 2 cm of the esophagogastric junction on the lesser curvature and/or posterior wall of the cardia. This represents 11.7% (9/77) of the cases of cancer at the esophagogastric junction. This incidence contrasts with the 39.5% of early cancers elsewhere in the stomach. Overall, 36.6% (479/1308) were early gastric cancer. Eight of these 9 patients were resected through the abdominal approach and one by the thoracoabdominal approach. Seven underwent proximal and 2 underwent total gastrectomy. On microscopic examination, one cancer was m-cancer, and 8 were sm-cancer. Lymphnode metastasis was found to be absent in all 9 cases. The prognosis of cancer of the cardia is generally poorer than that for adenocarcinoma in the corpus or distal stomach. However, cancer recurrence 22-91 months postoperatively was zero in these 9 cases of early gastric cancer at the esophagogastric junction.

The results of the surgical resection of the paraaortic lymph nodes metastases of the gastric cancer were evaluated in 116 cases. The frequency of the dissection of paraaortic lymph nodes (No. 16 lymph node) was 20%; the metastatic ratio was 24%. When classified by regions, the highest frequency of metastasis was observed in the cases of upper gastric cancer. The effect of the dissection of No. 16 was more conspicuous when No. 16 metastases were absent than when No. 16 metastases were present. Particularly, the prognosis was improved in the case of n1 and n2 population of the cases which were positive with respect to the proximal lymph node metastasis. It appears altogether that the dissection has considerable significance as prophylactic treatment.

A 27-year-old female was admitted with fever and abdominal pain. She was diagnosed as having a tumor above the right kidney by computed tomoraphy. No endocrinological abnormality was recognized, so surgical removal of the tumor was performed. A cut surface of the tumor was yelowisch and revealed thin capsules. The histopathologcal diagnosis was a ganglioneuroma. Retroperitoneal ganglioneuroma is rare and only 62 cases including this case have been reported in Japan. The disease was predominant in females and patients between 2 and 20 years old were common. Abdominal pain and swelling were the main clinical symptoms. High values of VMA, HVA and VIP were observed in 11 of 12 patients youner than 6 years old. In all the cases, a strong concern with neuroblastoma or ganglioneuroblastoma was suggested. There were no cases diagnosed exactly before operation. The necessity of correct diagnosis for a surgical specimen was emphasized. Surgical treatment was performed in all cases, leading to satisfactory prognoses to them except one which showed a fatal recurrence.

We previously demonstrated that the antitumor efficacy of various antitumor agents such as 5-fluorouracil and cisplatin against experimental solid tumors was enhanced by pre- or simultaneous administration of human epidermal growth factor (hEGF). In the present study, the combined therapy by hEGF and mitomycin C (MMC) as an antitumor agent was studied in A431 solid tumor-bearing mice to determine the dosage schedule of hEGF. When MMC alone was injected intraperitoneally (2 mg/kg) every 7th day to the tumor-bearing mice, tumor weights increased to 2138±285 mg from 282±41 mg during 22 d. Tumor weight in every day treatment of hEGF alone for 21 d increased to the same extent in the treatment by MMC alone. On the other hand, the increase of the solid tumor weight in the every day treatment and in the every 7th day treatment of hEGF, in combination with the every 7th day administration of MMC, were as follows ; from 282±41 mg to 1522±357 mg (71.2±16.7% of MMC alone) and from 280±44 mg to 1245±150 mg (58.2±7.0% of MMC alone), respectively, demonstrating a greater antitumor potency of MMC in the combination with the every 7th day treatment of hEGF. Both combined therapies did not affect the toxicity of MMC as evaluated by decrease in nontumorous body weight. Single subcutaneous administration of hEGF to A431 tumor-bearing mice caused the decrease of the binding capacity of hEGF to A431 tumor cells by 80% 24 h after the administration. However, the decreased binding capacity recovered to the untreated control level 4 d after the administration of hEGF. In conclusion, the every 7th day treatment of hEGF was superior than the every day treatment in its function to increase the susceptibility of A431 solid tumor to MMC.

There are very few studies on the interaction between dental alloys and oral tissues. The effect of direct contact with copper (Cu) on the cellular function of human gingival fibroblast (HGF) derived from the periodontal tissues was investigated. When HGF cells were inoculated onto a Cu plate, the viability of HGF cells immediately declined. This was accompanied by vacuolization and chromatin condensation near the nuclear membrane. The intracellular concentration of spermidine and spermine declined, whereas that of putrescine slightly increased. Amino acid analysis of the medium revealed that glutamine was consumed at the greatest rate, amounting to more than half of the total amino acid consumption. Contact with the Cu plate resulted in the complete elimination of glutamine utilization and a simultaneous increase in the production of most amino acids, possibly due to enhanced proteolysis. This was accompanied by a time-dependent increase in the consumption of cystine, possibly due to oxidative reactions, and the enhanced production of glycine and glutamic acid. These data suggest that the contact with the Cu plate induced non-apoptotic cell death in HGF cells, which was tightly coupled with a rapid dysfunction of amino acid and polyamine metabolism.

Most previous studies of the induction of tumor cell apoptosis by morphine have been conducted with concentrations very much higher than those used clinically. An investigation of the ability of morphine to induce apoptosis at its clinical concentration (10(-8) M) was therefore undertaken. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, induction of early apoptosis and necrosis by fluorescence-activated cell sorter (FACS) analysis with Annexin V and propidium iodide (PI), activation of caspase -2, -3, -8 and -9 by cleavage of specific substrates, DNA fragmentation by agarose gel electrophoresis, radical intensity and O2- scavenging activity by ESR spectroscopy. Millimolar concentrations of morphine showed higher cytotoxicity against human tumor cell lines (HL-60, A549, MCF7) than against normal human cells (HGF, HPC, HPLF). The clinical concentration of morphine produced early apoptotic markers in HL-60 and A549 cells whereas it induced higher numbers of necrotic cells in MCF7 cells, both in a naloxone-sensitive manner. The clinical concentration of morphine failed to activate any caspase species and induced only trace amounts of internucleosomal DNA fragmentation, in contrast to cytotoxic concentrations of morphine. Morphine, with a C-3 hydroxyl group, showed higher cytotoxicity and O2- scavenging activity than codeine, in which the hydroxyl group at C-3 was replaced with a methoxy group, suggesting the involvement of a radical-mediated reaction. The present report may offer new strategies for treatment and prevention of cancer using a clinical concentration of morphine not only as an anti-nociceptive, but also as an apoptosis or necrosis inducer.

Twenty trihaloacetylazulene derivatives with one atom of fluorine, chlorine, bromine or iodine was investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast, HGF; pulp cell, HPC; periodontal ligament fibroblast, HPLF) and four human tumor cell lines (squamous cell carcinoma, HSC-2, HSC-3, HSC-4; promyelocytic leukemia, HL-60). There was no apparent difference in the cytotoxic activity between 2-methoxyazulenes [1a-1e, 2a-2e] and 2-ethoxyazulenes [3a-3e, 4a-4e]. Trichloroacetylazulenes [2a-2e, 4a-4e] generally showed higher cytotoxicity and tumor-specificity (expressed as a TS value) as compared with the corresponding trifluoroacetylazulenes [1a-1e, 3a-3e]. Substitution of chloride [1c, 2c, 3c. 4c], bromide [1d, 2d, 3d, 4d] or iodine [1e, 2e, 3e, 4e] at the C-3 position further enhanced cytotoxic activity against four tumor cell lines, especially HL-60 cells. Among twenty trihaloacetylazulene derivatives, two compounds [2d] and [4c] showed the highest tumor specificity (TS =3.5 and2.5, respectively). Compounds [2d] and [4c] induced apoptotic cell death characterized by caspase-3, -8 and -9 activation and internucleosomal DNA fragmentation in HL-60 cells. On the other hand, compounds [2d] and [4c] induced autophagic cell death characterized by lower activation of caspases, lack of DNA fragmentation, vacuolization and autophagosome formation detected by acridine orange and LC3-GFP fluorescence, without the decline of the intracellular concentration of three major polyamines in HSC-4 cells. The cytotoxic activity of [4c], but not [2d], was slightly reduced by 3-methyladenine, an inhibitor of autophagy. These results suggest the diversity of cell death type induced in human tumor cell lines by trihaloacetylazulene derivatives.

Rikko-san (RKS) and its ingredients were investigated for their activity to stimulate prostaglandin E2 (PGE2) production by unstimulated and lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells. LPS significantly stimulated the production and extracellular secretion of PGE2 by Raw 264.7 cells. RKS dose-dependently modified the LPS-stimulated PGE2 production. A lower concentration (0.04-0.4 mg/ml) of RKS stimulated PGE2 production without or with LPS, whereas a higher concentration (4 mg/ml) of RKS inhibited the LPS-stimulated PGE2 production, suggesting the presence of both stimulator(s) and inhibitor(s) for PGE2 production. The inhibitory effect of a higher concentration of RKS was slightly neutralized by supplementation of exogenous arachidonic acid. Among five RKS ingredients, Kanzo inhibited the LPS-stimulated PGE2 production to the greatest extent, whereas other ingredients (Shoma, Ryutan, Saishin, Bofu) stimulated PGE2 production. Western blot analysis demonstrated that RKS and all its ingredients enhanced the intracellular concentration of cyclooxygenase-2, whereas RKS did not significantly affect the phospholipase A2 protein level. The present study demonstrates that the concentration-dependent effect of RKS on the PGE2 production by macrophage cannot be explained only by the expression levels of cyclooxygenase-2 or phospholipase A2.

This study investigated the impact of single, double, and triple insecticide sprayings on indoor infestation of Triatoma dimidiata in Jutiapa, Guatemala. Up to three successive insecticide sprayings were applied in villages, where the indoor infestation index was > 5% before each spraying round or located adjacent to the persistently infested villages. Among 64 villages with single spraying, the mean indoor infestation index reduced from 20.8% to 1.4% after 12 months, but rose to 8.1% after 33 months. In 40 double-sprayed villages, it decreased from 41.9% to 11.9% by the first spraying and to 4.8% by the second spraying. For 12 villages with triple spraying, it reduced from 40.6% to 13.2%, 10.9%, and 4.1% through each spraying round. Geographic analysis showed that originally highly infested villages were spatially clustered and were likely to remain infested after the sprayings. Indoor infestation of T. dimidiata can be controlled with less than three rounds of spraying.

The effects of 26 trihaloacetylazulene derivatives on nitric oxide (NO) production by the mouse macrophage-like Raw 264.7 cells was investigated. The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared with the corresponding trifluoroacetylazulenes [1a-13a]. All the compounds inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. 3-Trifluoroacetylguaiazulene [8a], 1-trifluoroacetyl-4,6,8-trimethylazulene [10a], 3-methyl-l-trichloroacetylazulene [2b] and 3-ethyl-1-trichloroacetylazulene [3b] showed lower cytotoxic activity and most effectively inhibited NO production. Western blot analysis revealed that compounds [8a, 1Oal dose-dependently reduced the intracellular concentration of inducible NO synthase (iNOS), whereas compounds [2b, 3b] only marginally affected the iNOS protein expression. RT-PCR analysis showed that compounds [8a, 2b] reduced the iNOS mRNA expression by approximately 50%. These compounds affected cyclooxygenase-2 protein and mRNA expression, depending on the concentrations. ESR spectroscopy revealed that compounds [8a, 10a, 2b, 3b] neither produced radical, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study showed the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages.

The effects of combined beta-cryptoxanthin and zinc on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vivo were investigated. Rats were orally administered either vehicle, beta-cryptoxanthin (5 or 10 microg/100 g body weight), zinc sulfate (0.1 or 0.5 mg Zn/100 g), or their combination once a day for 7 d. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal tissues was not significantly altered by the administration of beta-cryptoxanthin (5 microg/100 g) or zinc (0.1 or 0.5 mg/100 g). Combined administration of beta-cryptoxanthin (5 microg/100 g) and zinc (0.1 or 0.5 mg/100 g) caused a synergistic increase in calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal tissues. The effect of beta-cryptoxanthin (5 or 10 microg/100 g) in increasing calcium and DNA contents in the metaphyseal tissues was significantly enhanced by the combined administration of zinc (0.1 or 0.5 mg/100 g), but did not have a significant effect on the metaphyseal components. The metaphyseal alkaline phosphatase activity was markedly increased by the combination of beta-cryptoxanthin (5 microg/100 g) and zinc (0.1 or 0.5 mg/100 g). This study demonstrates that the oral administration of the combination of zinc at lower doses synergistically enhances beta-cryptoxanthin-induced anabolic effects on bone components in the femoral tissues of rats in vivo.