Evaluation of genotoxicity and subchronic toxicity of standardized rose hip extract.

Rose hip is the fruit of the rose plant, which is widely used in food, cosmetics and as a traditional medicine. Therefore, rose hip is considered safe and has a sufficient history of consumption as food. However, few studies have reported on the safety of rose hip extracts in toxicological analyses. Thus, to evaluate the safety of rosehip polyphenol MJ (RHPMJ), an aqueous ethanol extract standardized with the trans-tiliroside content, we performed genotoxicity and 90-day repeated oral dose toxicity studies in compliance with the Organisation for Economic Co-operation and Development-Good Laboratory Practice. RHPMJ did not induce gene mutations in reverse mutation tests of Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains and did not induce chromosomal aberrations in cultured Chinese hamster lung (CHL/IU) cells. Moreover, micronucleus tests using rat bone marrow showed RHPMJ had no micronucleus-inducing potential. Finally, 90-day repeated oral dose toxicity studies (100-1000 mg/kg) in male and female rats showed no treatment-related toxicity in rats. These data indicate that the RHPMJ had no genotoxicity and a no-observed-adverse-effect level greater than 1000 mg/kg in rats.

Roles of prostaglandin E receptors in mesangial cells under high-glucose conditions.

BACKGROUND: High glucose reportedly stimulates prostaglandin (PG) E2 production and DNA synthesis in mesangial cells (MCs). However, the pathophysiological significance of PGE2 in MCs has remained unclear. METHODS: The effects of prostanoids on [3H]-thymidine uptake and cAMP production in rat MCs cultured with 5.6 mM glucose, 25 mM glucose, or 5.6 mM glucose supplemented with 19.4 mM mannitol were examined. The gene expression of PGE2 receptor (EP) subtypes in MCs was analyzed with Northern blotting techniques. RESULTS: Northern blotting indicated EP1 and EP4 gene expression in MCs. EP1 agonists and PGE2 stimulated [3H]-thymidine uptake in MCs. EP1 antagonists dose dependently attenuated high-glucose-induced [3H]-thymidine uptake, which suggests EP1 involvement, by an increase in intracellular Ca2+, in DNA synthesis of MCs. On the other hand, forskolin, db-cAMP, and 11-deoxy-PGE1, an EP4/EP3/EP2 agonist, significantly decreased DNA synthesis in MCs. These inhibitory effects are thought to be mediated via EP4 as a result of an increase in cAMP synthesis. The effects via EP4 seem to be particularly important because PGE2-induced cAMP synthesis was significantly attenuated in the high-glucose group compared with the mannitol group, in which [3H]-thymidine uptake did not increase in spite of augmented PGE2 production. CONCLUSION: The increase in DNA synthesis in MCs under high-glucose conditions can be explained, at least in part, by the high-glucose-induced inhibition of cAMP production via EP4, which augments EP1 function in conjunction with the overproduction of PGE2.

[Preliminary evaluation of adjuvant chemotherapy against stage Ib or II gastric cancer].

The prognostic influence of post-operative adjuvant chemotherapy on stage I b or II gastric cancer was studied retrospectively. The immunohistochemical expressions of p53 protein and thymidine phosphorylase (TP) were also examined; the relations between these protein expressions and clinicopathological features along with the effect of adjuvant chemotherapy were also investigated. The 5-year survival rate of the patients who received adjuvant chemotherapy was 95.5%, which was better than that (89.8%) of those who did not, although the difference did not reach significance (p = 0.09). The venous invasion of tumor was slight frequently observed in p53 or TP positive cases than negative cases, respectively (p < 0.1), but no significant associations were found between the t-, n- or ly-factor, and p53 or TP expression. Moreover p53 and TP expression had no significant influence on post-operative survival. But, among the patients with p53- or TP-positive tumor, adjuvant chemotherapy conferred survival benefits, although the difference did not reach significance. The 5-year survival rate was 100% with adjuvant chemotherapy, 84.3% without chemotherapy in p53-positive patients (p = 0.137), 97.0% with adjuvant chemotherapy, and 90.8% without chemotherapy in TP-positive tumors (p = 0.326).

Immunocytochemical localization of prostaglandin EP3 receptor in the rat hypothalamus.

A rabbit antibody against an N-terminal portion of rat prostaglandin EP3 receptor (EP3R) was produced to examine the distribution of EP3R in the rat hypothalamus. The antibody specifically recognized EP3R proteins in rat brain extract, in membrane fractions of rat kidney, and in membrane fractions of EP3R-expressing culture cells. Intense EP3R-like immunoreactivity was observed in the median preoptic nucleus, medial preoptic area, parastrial nucleus, compact part of the dorsomedial hypothalamic nucleus, and dorsal part of the premammillary nucleus. These results suggest that prostaglandin E2 mediates various actions in the hypothalamus, such as fever induction in the preoptic area, through EP3R.

Copresence of prostaglandin EP2 and EP3 receptors on gastric enterochromaffin-like cell carcinoid in African rodents.

BACKGROUND & AIMS: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis, which might be a useful model of normal ECL cells. METHODS: A [3H]PGE2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP2 and EP3 receptor complementary DNAs was conducted. RESULTS: [3H]PGE2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE1 = PGE2 > enprostil > PGF2 alpha. Although PGE1 and PGE2 stimulated 5'-cyclic adenosine monophosphate (cAMP) production, neither PGF2 alpha nor enprostil had any effect. On the other hand, all of PGE1, PGE2, PGF2 alpha, and enprostil attenuated the forskolin-induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP2 and EP3 receptor messenger RNAs was confirmed by RNA blot analysis. CONCLUSIONS: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP2 linked to cAMP production and EP3 coupled with inhibitory guanosine 5'-triphosphate-binding proteins mediating the inhibition of cAMP production.

Mouse prostaglandin E receptor EP3 subtype mediates calcium signals via Gi in cDNA-transfected Chinese hamster ovary cells.

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.

Gastrin receptor genes are expressed in gastric parietal and enterochromaffin-like cells of Mastomys natalensis.

Although gastric enterochromaffin-like (ECL) carcinoid tumors are known to develop in patients with long-standing hypergastrinemia, the expression of the gastrin receptor gene in ECL cells has not yet been demonstrated. Therefore, this study was designed to examine gastrin receptor gene expression in ECL cells. Mastomys gastric mucosal cells isolated by enzyme dispersion were separated into 10 fractions (F1-10) by centrifugal elutriation. Each fraction was examined histologically to determine whether they contained ECL and/or parietal cells and Northern blot analysis was used to confirm the presence of histidine decarboxylase and H+, K(+)-ATPase gene expression. ECL cells were found only in fractions 1 and 2, whereas parietal cells were detected in fractions 6-10. Gastrin receptor gene expression was demonstrated in both parietal cell-rich and ECL cell-rich fractions. In addition, the gastrin receptor cDNA sequences obtained from the two of the fractions (F1 and 8) were identical. These results suggest that gastrin receptor genes are expressed in ECL cells as well as in parietal cells and that these receptors are identical.

Cloning and expression of a cDNA for rat prostaglandin F2 alpha receptor.

We have cloned a cDNA for rat prostaglandin (PG) F2 alpha receptor from cultured rat astrocytes. The cDNA encodes a polypeptide of 366 amino acids with seven putative transmembrane domains. Specific binding of [3H]PGF2 alpha in membranes of COS-7 cells transfected with the cDNA was displaced with unlabeled PGs in the order of PGF2 alpha > PGD2 > PGE2 > PGI2. In the cDNA-transfected LLC-PK1 cells, PGF2 alpha stimulated phosphoinositide hydrolysis. A significant 4.7-kb mRNA transcript was detected in cultured rat astrocytes and whole brain and pregnant ovary of adult rats by Northern blot analysis.

Cloning and expression of a cDNA for the human prostacyclin receptor.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost >> carbacyclin > prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF 2 alpha did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

cDNA cloning of a mouse prostacyclin receptor. Multiple signaling pathways and expression in thymic medulla.

A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.

Cloning and expression of a cDNA for mouse prostaglandin F receptor.

A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.

Effect of the dietary alpha-linolenate/linoleate balance on leukotriene production and histamine release in rats.

Rats were fed semi-purified diets supplemented either with safflower seed oil rich in linoleate (18:2n-6) or with perilla seed oil rich in alpha-linolenate (18:3n-3) through two generations. In the major phospholipids of polymorphonuclear leukocytes (PMNs), the proportions of n-6 polyunsaturated fatty acids (18:2, 20:4, 22:4 and 22:5) were higher but those of n-3 acids (20:5, 22:5 and 22:6) were lower in the safflower group than in the perilla group. When stimulated with a calcium ionophore, the PMNs from the safflower group produced 27% more leukotriene (LT)B4 than those from the perilla group. The formation of LTB5 which has biological activities less than 1/10 those of LTB4, was negligible in the safflower group but was 40 ng/10(7) PMN cells in the perilla group. The amount of the total LTB formed in the perilla group tended to be more than in the safflower group. The formation of SRS-A (slow-reacting substances of anaphylaxis) by PMNs was determined by measuring the spasmogenic activities of LTs on guinea pig ileum. SRS-A activity was 59% higher in the safflower group than in the perilla group. In contrast, histamine release from rat peritoneal mast cells was not significantly different between the two groups. Thus, the increasing the alpha-linolenate/linoleate ratio of diets results in the decreased formation of LTs derived from 20:4n-6 in PMNs. This may be beneficial in lowering the severity of allergic and inflammatory responses caused by LTs, and thereby shifting the pathological symptoms to normal self-defense mechanism.

Reactivation mechanisms of thiamine with thermostable factors.

It was observed, in vitro, that the water extract of the fermented-tea customarily chewed by Thai people has a similar thermostable thiamine-inactivating factor to that found in the water extract of fern. It was also observed that the percentage of thiamine disulfide formed from thiamine with some flavones, catechol, pyrogallol, caffeic acid, dihydroxyphenylalanine, and hemin is greater at pH 7.5 than at pH 7.0. With some flavonoids, such as quercetin, rutin, and 6,7,4'-trihydroxyisoflavone, and pyrogallol, hemin, catechol and caffeic acid at pH 7.5, around 30-100% of thiamine is changed into thiamine disulfide. Water extract of shiitake, okra, coffee, black tea and fukinoto have only weak activities of thermostable thiamine-inactivating factors as a large percentage of thiamine disulfide is formed from thiamine even at pH 7.0. 2-Methyl-4-amino-5-aminomethylpyrimidine was isolated from the reaction mixture of 1 g thiamine with 20 mg catechol (1:0.5 mole) at pH 7.0, 45 degrees C, and identified with the synthesized pyrimidine.