Periodontal pathogen Aggregatibacter actinomycetemcomitans LPS induces mitochondria-dependent-apoptosis in human placental trophoblasts.

OBJECTIVE: Increasing evidence suggests an association between periodontal disease and low birthweight (LBW); however the underlying molecular mechanisms are yet to be fully elucidated. In this study, we performed a microarray analysis to observe the human placental trophoblast-like BeWo cells response to lipopolysaccharide (LPS) from periodontopathogen Aggregatibacter actinomycetemcomitans (Aa), in order to investigate the molecular basis of mechanisms for periodontitis-associated LBW. In vivo pregnant rats were also used to confirm the in vitro results. STUDY DESIGN: The effects of Aa-LPS on cultured human placental trophoblast-like BeWo cells were studied using a DNA microarray, Ingenuity Pathway Analysis, real-time PCR and poly-caspase staining. The in vivo effects of Aa-LPS in pregnant rats were examined using TUNEL assays. RESULTS: In BeWo cells, Aa-LPS increased levels of cytochrome c, caspase 2, caspase 3, caspase 9 and BCL2-antagonist/killer 1 mRNA, decreased those of B-cell CLL/lymphoma 2, BCL2-like 1 and catalase mRNA and increased poly-caspase activity, all of which are consistent with activation of the mitochondria-dependent apoptotic pathway. TUNEL assays confirmed the increased incidence of apoptosis in placentas of Aa-LPS-treated rats (p < 0.001). CONCLUSION: Aa-LPS induces apoptosis in human trophoblasts via the mitochondria-dependent pathway, and this effect may contribute to the pathogenesis of periodontitis-associated LBW.

MCP-1 production in temporomandibular joint inflammation.

Synovitis, which is characterized by the infiltration of inflammatory cells, often accompanies progression of temporomandibular joint disorder (TMD) symptoms. Because IL-1ß is elevated in synovial fluids obtained from TMDs, we hypothesized that IL-1ß-responsive genes in synoviocytes may help identify the putative genes associated with synovitis. Using microarray analysis, we found that monocyte chemoattractant protein-1 (MCP-1) mRNA levels were elevated in IL-1ß-stimulated synoviocytes. MCP-1 is a member of the chemokine superfamily. The production of MCP-1 was increased in synoviocytes treated with IL-1ß. When IL-1ß was injected into the cavities of rat TMJs, inflammatory cells and MCP-1-positive cells were detected in the synovial tissues. Furthermore, MCP-1 levels were higher in synovial fluids from individuals with pain compared with those without pain. Inhibitors of MAP-kinases and NF-κB reduced IL-1ß-induced MCP-1 production. These results suggest that MCP-1 stimulated by IL-1ß is one of the factors associated with the inflammatory progression of TMDs.

Expression of cyclooxygenase-1 and -2 in IL-1beta-induced synovitis of the temporomandibular joint.

BACKGROUND: In this study, we analyzed the gene expression profile of fibroblast-like synoviocyte (FLS) cultures from the temporomandibular joint (TMJ) to identify candidate genes associated with intracapsular pathologic conditions of TMJ. Cyclooxygenase (COX)-2 was one of the genes in FLS upregulated following stimulation by interleukin (IL)-1beta, a cytokine thought to play a key role in several pathological conditions. This study investigated the expression of COX-1 and COX-2 in cultured human FLS and rat TMJ synovium following stimulation with IL-1beta. METHODS: RNA was isolated from human FLS after IL-1beta treatment. COX-1 and -2 expression was examined using a GeneChip and real-time polymerase chain reaction. Prostaglandin E(2) (PGE(2)) levels in conditioned media from FLS were measured using enzyme-linked immunosorbent assay. Synovial tissues from TMJs of IL-1beta-injected rats were examined for COX-1 and COX-2 expression by immunohistochemical staining. RESULTS: Following treatment of FLS with IL-1beta, expression of the COX-2 gene increased up to 8 h and peaked at 4 h, whereas COX-1 expression did not change. Stimulation with IL-1beta increased the level of PGE(2) in conditioned media of cultured FLS in a time-dependent manner up to 48 h. Immunohistochemistry showed a strong positive staining for COX-2 in the lining and sub-lining synovial tissues of the TMJ of IL-1beta-injected rats. In contrast, staining for COX-1 was the same in synovial tissues with and without IL-1beta injection. CONCLUSION: These data suggest that COX-2 expression stimulated by IL-1beta stimulates the production of PGE(2) in FLS and plays important roles in the progression of inflammation in TMJ.

Cerebral cortex activity during supramaximal exhaustive exercise.

AIM: The purpose of this study was to examine the effect of fatigue resulting from supramaximal dynamic exercise on cerebral cortex activity. METHODS: Five healthy male subjects (age 24.6+/-0.4 years, body weight 62.9+/-1.1 kg, height 175.3+/-1.2 cm, and maximal O2 uptake per body mass 48.4+/-1.3 ml/kg/min) participated in this study. All subjects performed at 120% of maximal oxygen uptake (VO2peak) on a cycle ergometer until reaching a state of volitional fatigue. Cerebral oxygenation was measured by near-infrared spectroscopy (NIRS) throughout the supramaximal constant exhaustive exercise. RESULTS: The mean exercise duration of the subjects was 147.2+/-3.4 s. The peak value of blood lactate concentration within 3-10 min after the exercise test was 14.4+/-0.1 mmol/l. Cerebral oxygenation (8.8+/-1.8 micromol/l) was increased significantly during the first minutes of exercise compared with the pre-exercise value (p<0.05) and cerebral oxygenation decreased with the passage of time during exercise. Cerebral oxygenation at the end of exercise decreased significantly compared with the resting value (-29.9+/-3.4 micromol/l, p<0.05). CONCLUSION: These findings suggest that the exhaustive exercise induces the decrease of cerebral function and that the fatigue resulting from dynamic exercise decreases the cerebral cortex activity.

Photosynthetic electron transport inhibition by pyrimidines and pyridines substituted with benzylamino, methyl and trifluoromethyl groups.

The decrease of the number of ring nitrogen atoms of 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines on herbicidal activity and inhibition of photosynthetic electron transport (PET) was assayed using thylakoids from Spinacia oleracea or atrazine-resistant Chenopodium album. Three 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines, nine pyrimidines with a benzylamino-, methyl- and trifluoromethyl-group, 2-benzylamino-6-methyl-4-trifluoromethyl- pyridine and N-benzyl-3-methyl-5-trifluoromethylaniline were synthesized and assayed. 2-(4-Bromobenzylamino)-4-methyl-6-trifluoromethylpyrimidine exhibited the highest PET inhibitory activity against Spinacia oleracea thylakoids of all compounds tested. The 2-benzylaminopyrimidines and 2-methylpyrimidines having a 4-halobenzylamino group exhibited higher PET inhibition than atrazine and 2-trifluoromethylpyrimidines against Spinacia oleracea thylakoids. These PET inhibitory active compounds also exhibited a strong and similar inhibition both against atrazine-resistant Chenopodium album thylakoids as well as against thylakoids from wild-type Chenopodium. The herbicidal activity of 4-(4-bromobenzylamino)-2-methyl-6- trifluoromethylpyrimidine was equivalent to that of known herbicides like simetryne, simazine or atrazine.

[Characteristics of auricular changes by ageing--a case report from Saga Prefecture].

Measurements of auricles were done about 317 volunteers living in the Saga Prefecture at the age of 50 to 94 years old (71 men and 246 women), from July until November, 1999. 1) A growing tendency was observed in both male and female physiognomic ear length. 2) In contrast, there was no significant increase or decrease in the ear width. 3) A decreasing tendency was observed in the ear index of both male and female volunteers.

In vitro pharmacological profile of SK-896, a new human motilin analogue.

SK-896 ([Leu(13)]motilin-Hse) is a new human motilin analogue synthesized by Escherichia coli using a biotechnological method. We investigated the binding of SK-896 to motilin receptors and the contractile effect of SK-896 on smooth muscle preparations isolated from the gastrointestinal tract and various regional organs in order to clarify its in vitro pharmacological profile. SK-896 inhibited the binding of (125)I-human motilin to rabbit gastroduodenal motilin receptors with the same potency as unlabeled human motilin. The IC(50) values of SK-896 and human motilin were 3.5 +/- 1.5 and 3.1 +/- 1.8 nmol/l, respectively. The K(d) of human motilin was 3.0 +/- 1.5 nmol/l, and the Ki of SK-896 was 3.4 +/- 1.5 nmol/l. SK-896 induced contraction of smooth muscle preparations isolated from rabbit duodenum in a concentration-dependent manner. However, there was no effect of SK-896 on duodenal preparations isolated from the dog and the rat. SK-896 thus exhibited species specificity in its contractile effect. We next investigated the effect of SK-896 on various smooth muscle preparations isolated from rabbit gastrointestinal tract, trachea, bladder, gallbladder, uterus, vas deferens and artery. Results showed that SK-896 induced contraction of smooth muscle preparations isolated from gastrointestinal tract, with potencies in the order duodenum > gastric pylorus = jejunum = descending colon > ascending colon >/= ileum. However, there was no effect of SK-896 on smooth muscle preparations from gastric fundus and other regional organs. SK-896 thus exhibited regional specificity in its contractile effect. Moreover, the effects of SK-896 on smooth muscle preparations from rabbit duodenum were the same as those of human motilin, and were not inhibited by pretreatment with tetrodotoxin and atropine but were inhibited by verapamil. These findings indicate that SK-896 has the same pharmacological profile as human motilin. They suggest that SK-896 acts on gastrointestinal smooth muscle isolated from rabbit directly and specifically.

Mode of action of novel 2-(benzylamino)-4-methyl-6-(trifluoro- methyl)-1,3,5-triazine herbicides: inhibition of photosynthetic electron transport and binding studies.

Novel 2-(benzylamino)-4-methyl-6-(trifluoromethyl)-1,3,5-triazines have the same 1,3,5-triazine skeleton as atrazine, although some of them, for example, 2-(3-chlorobenzylamino)-4-methyl-6-(trifluoromethyl)-1,3,5-tria zin e [pI(50)(spinach) = 7.21], show a >3 times stronger photosynthetic electron transport inhibitory activity than atrazine [pI(50)(spinach) = 6.72]. The new triazines have only one amino group at the triazine ring, and their molecular shapes are different from atrazine. The replacement of the bound [(14)C]atrazine by 1,3,5-triazines was tested to determine whether the novel 1,3,5-triazine analogues exhibit the same binding pattern at the D1-protein as atrazine. It was found that [(14)C]atrazine bound to the D1-protein was replaced by the triazine tested by a clearly competitive interaction. Obviously, the novel 1,3,5-triazines are attached to the same binding niche as atrazine.

Nicotinamide inhibits sodium-dependent phosphate cotransport activity in rat small intestine.

BACKGROUND: We recently reported that the administration of niceritorol (a nicotinic acid derivative which improves lipid metabolism and peripheral circulation, and is used for the treatment of hyperlipidaemia and impaired peripheral circulation) to patients with hyperphosphataemia undergoing dialysis decreased the serum phosphate (Pi) concentration. We found that this was due to an acceleration of faecal Pi excretion by niceritrol. METHODS: Intestinal brush border membrane vesicles (BBMVs) were prepared from rat jejunum, and the Na+-dependent and Na+-independent Pi transport activities in these vesicles were measured. In addition, the functional Pi transporter from rat small intestine was injected in Xenopus oocytes, and the effect of nicotinamide on the levels of its expression were measured by northern blotting. RESULTS: The Na+-dependent component was significantly decreased in the BBMVs isolated from rats treated with nicotinamide, while the Na+-independent component was not changed. Kinetic studies demonstrated that the decreased activity was due to reduction of the Vmax value and not an elevation of the Km values. When poly(A)+RNA from rats treated with nicotinamide was microinjected into Xenopus oocytes, the Pi transport activity was significantly decreased compared with that in the control animals. In addition, there were no significant changes in Na/Pi cotransporters and activators, but the vitamin D receptor mRNA level was reduced to 80% of the control level. CONCLUSIONS: These observations suggest that nicotinamide may regulate the expression of a major functional Na/Pi cotransporter in the rat small intestine.

Effects of niceritrol on faecal and urinary phosphate excretion in normal rats.

BACKGROUND: Phosphate binders such as aluminium hydroxide and calcium carbonate have several side-effects so that they are not ideal for clinical use. Recently we reported that administration of niceritrol at 750 mg/day to patients with HDL hypocholesterolaemia undergoing dialysis decreased the serum phosphate concentration. To clarify the mechanism of reduction of the serum phosphate concentration by niceritrol in patients undergoing dialysis, the effects of niceritrol on faecal and urinary phosphate excretion were examined in normal rats. METHODS: Niceritrol suspension in 5% gum arabic was administered for 4 days in normal rats. Faeces and urine were collected in metabolic cage and analysed for phosphate content. RESULTS AND CONCLUSIONS: Faecal phosphate excretion significantly increased in the groups with administration of 100 and 500 mg/kg niceritrol, but not in the group with 20 mg/kg. On the other hand, urinary phosphate excretion was not significantly changed in the groups on 20-500 mg/kg. Retention of phosphate was significantly suppressed in the groups on 100 and 500 mg/kg. A increased faecal phosphate excretion by niceritrol is presumably the mechanism by which serum phosphate concentration is reduced in dialysis patients.

Complex blockade of TTX-resistant Na+ currents by lidocaine and bupivacaine reduce firing frequency in DRG neurons.

Mechanisms of blockade of tetrodotoxin-resistant (TTXr) Na+ channels by local anesthetics in comparison with the sensitivity of tetrodotoxin-sensitive (TTXs) Na+ channels were studied by means of the patch-clamp technique in neurons of dorsal root ganglions (DRG) of rat. Half-maximum inhibitory concentration (IC50) for the tonic block of TTXr Na+ currents by lidocaine was 210 micromol/l, whereas TTXs Na+ currents showed five times lower IC50 of 42 micromol/l. Bupivacaine blocked TTXr and TTXs Na+ currents more potently with IC50 of 32 and 13 micromol/l, respectively. In the inactivated state, TTXr Na+ channel block by lidocaine showed higher sensitivities (IC50 = 60 micromol/l) than in the resting state underlying tonic blockade. The time constant tau1 of recovery of TTXr Na+ channels from inactivation at -80 mV was slowed from 2 to 5 ms after addition of 10 micromol/l bupivacaine, whereas the tau2 value of approximately 500 ms remained unchanged. The use-dependent block of TTXr Na+ channels led to a progressive reduction of current amplitudes with increasing frequency of stimulation, which was

Determination of the absolute configuration of urothion.

In order to determine the absolute configuration of urothion (1), the CD spectra of tri-4-chlorobenzoyl derivatives of 2-amino-6-[(3R)-3,4-dihydroxybutyl]pteridin-4(3H)-one and its (3S) compound were compared with that of the tri-4-chlorobenzoyl derivative obtained from the natural product. R-Configuration was concluded for the secondary hydroxyl group on the side chain of 1, which is the same configuration as that of molybdopterin (2). This supports the view that 1 might be a urinary metabolite of 2.

Influence of diet composition and malocclusion on masticatory organs in rats.

The present study was undertaken to determine the effects of different dietary consistencies and malocclusion induced by extraction of molar teeth on the masticatory organs of weaning and adult rats, by determining the biochemical properties of masseter muscle, and also Ca and P levels in mandibular bone. Male SD rats, 3 and 20 weeks old, were divided into 3 groups. Group one (G-1) was maintained on a solid diet, and Groups two (G-2) and three (G-3) on a semi-solid diet. Furthermore, the mandibular molar teeth of G-3 rats were extracted. The experimental period was 120 days. The masseter muscle and mandibular bone weights of G-1 in weaning rats were increased significantly in comparison with G-3, but not in adult rats. The CPK activities in weaning and adult rats of G-1 were higher than those in the other two groups. The order of LDH activity in weaning and adult rats was G-3 > G-2 > G-1. G-2 and particularly G-3 showed significantly lower glycogen contents than G-1. The Ca and P contents of the mandibular bone in G-2 and G-3 were lower than those in G-1. These results suggest that a different dietary consistency and malocclusion induced by extraction of mandibular molar teeth have a considerable influence on the development of masticatory organs, mandibular bone and masseter muscle.

Effect of organic germanium compound (Ge-132) on experimental osteoporosis in rats.

1. The therapeutic effect of organic germanium compound, 2-carboxyethylgermaniumsesquioxide (Ge-132), for experimental osteoporosis was studied using ovariectomized rats maintained on a low calcium containing diet. 2. Serum calcitonin (sCT) level was decreased and serum parathyroid hormone (sPTH) level was increased by ovariectomy and the decrement and increment rates, respectively, were reduced by administration of Ge-132. Thus, the sCT/sPTH ratio was greater in the groups given Ge-132, indicating that the resorption was somehow inhibited by Ge-132. 3. The transverse strength of femur bone was significantly enhanced by Ge-132. 4. A trend was found in the group given Ge-132 for a larger femur cortical bone index. 5. The relative femur bone wet weight was greater in the group given Ge-132. 6. These results indicate that Ge-132 prevents decreased bone strength, and affects the femur cortical bone index, and bone mineral mass caused by osteoporosis.

Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal species.

An investigation was undertaken to determine whether analgesics and their derivatives (13 compounds), and anti-inflammatory drugs (4 compounds) had mutagenicity. Rec-assay was used to clarify specific DNA-damaging properties, and the Ames test was used to find back-mutations, using S-9 fractions obtained from the liver of 4 animal species pretreated with polychlorobiphenyl. In the Rec-assay, salicylic acid (2 mg), aspirin (5 mg), benzoic acid (4 mg), sulpyrine (0.4 mg), indomethacin (0.1 mg), oxyphenbutazone (0.1 mg) and diclofenac sodium (0.1 mg) showed a DNA-damaging tendency. In the Ames test, mutagenicity of methyl salicylate was demonstrated using the Salmonella typhimurium TA98 strain upon addition of hamster S-9 mixture. Weak mutagenicity was also found using the TA100 strain with rat S-9 mixture for salicylic acid, sulpyrine, indomethacin and oxyphenbutazone, and with hamster S-9 mixture for methyl salicylate, acetaminophen and phenacetine.

[Influence of sucrose administration on blood insulin level, and number of insulin receptor and affinity on isolated hepatocytes].

In order to investigate the process to form diabetic-like condition by giving continuously excessive amount of sucrose, rats were maintained with purified water (Purified Water Group, PWG) and 10% sucrose solution (Sucrose Group, SG), as drinking solution, ad libitum for 5 months, and were subjected to the following experiments: 1) Serum glucose (BG) and insulin level (IRI) in sucrose tolerance test (STT) were measured periodically up to 24 hours after administration. 2) The number and affinity of insulin receptor on isolated hepatocytes from the rats of PWG and SG were also investigated. At the termination of the experiment, 5 months after the initiation, the BG and IRI levels in SG were significantly higher compared to those of PWG. In STT, SG showed reduced glucose tolerance and also high blood insulin level. The number of insulin receptor of isolated hepatocytes of SG was approximately 23% lower than that of PWG and the affinity of SG was slightly lower than that of PWG. These results suggest that continuous high BG and IRI levels are indicated in the rats given large amount of sucrose continuously. The number of insulin receptor of such rat was also reduced by down regulation, resulting in an insulin-resistance. These cause further high blood glucose level and abnormality in metabolism, to result in a prediabetic impaired glucose tolerance.

Augmentation of wound healing by royal jelly (RJ) in streptozotocin-diabetic rats.

Chronically diabetic rats prepared by a single i.v. injection of streptozotocin were used to study whether royal jelly (RJ) possesses a hypoglycemic reaction and whether it can augment wound healing. Oral RJ administration of 10, 100 and 1000 mg/kg/day did not show any insulin-like activity (the hypoglycemic reaction). RJ, however, showed some anti-inflammatory activity by decreasing exudation and collagen formation in granulation tissue formation in the cotton pellet method. RJ also shortened the healing period of desquamated skin lesions. Thus, RJ possesses an anti-inflammatory action and is able to augment wound healing, but does not have an insulin-like action in streptozotocin-diabetic rats.

Concentrations of ampicillin in human serum and mixed saliva following a single oral administration of lenampicillin, and relationship between serum and mixed saliva concentrations.

The concentrations of ampicillin in serum and mixed saliva after a single oral administration of lenampicillin (500 mg) were determined by the paper disc method. The samples of serum and mixed saliva were obtained at 1, 2, 3 and 4 h after administration. The highest concentrations of ampicillin in serum and mixed saliva occurred 1 h after administration of lenampicillin, and were 11.55 and 0.060 micrograms/ml, respectively. A significant correlation coefficient between the concentrations of ampicillin in serum and mixed saliva, r = 0.71, P less than 0.001, was found.

Time-course study of gastric damages in rats by anti-inflammatory drugs using a gastroscope and its quantification.

Time-course studies on gastric damages in rats caused by nonsteroidal anti-inflammatory drugs (NSAIDs) were performed using a gastroscope, and the readings were quantified to obtain the Congestion-Hemorrhage Index (CHI) for evaluating the potencies of the damaging properties of NSAID. The correlation between CHI and Ulcer Index (UI), the quantified value obtained by the conventional methods, was highly significant at 6 and 24 hr after forced oral administration of NSAID. The peak CHIs of aspirin (300 mg/kg), indomethacin (60 mg/kg), mefenamic acid (300 mg/kg) and fenoprofen calcium (300 mg/kg) appeared approximately 24 hr after a single forced oral administration of drugs. Thus, it was suggested that an observation at 24 hr in addition to one at 6 to 7 hr might be necessary for the examination of damaged gastric mucosa. Under the present experimental conditions, fenoprofen calcium caused the greatest damages on gastric mucosa among the four NSAIDs. Mefenamic acid showed the least damaging potency on gastric mucosa, having a smaller CHI than that of aspirin. Indomethacin possessed a stronger damaging property than aspirin.

[Antitumor effects of royal jelly (RJ)].

Antitumor effects of Royal Jelly (RJ) were investigated employing the transplantable tumors of mouse advance leukemia L1210 and P388 strains and Ehrlich, Sarcoma-180 ascites and solid tumor strains. RJ was administered orally in a prophylactic-therapeutic (30 days before and 30 days after the transplantations of tumor cells) or a therapeutic (30 days after the transplantations of tumor cells) manner. Tumor cells were transplanted i.p. (ascites tumor) or s.c. (solid tumor). The daily dose of RJ was 0 (control), 10, 100, or 1000 mg/kg. In the case of the therapeutic experiments employing advance leukemia L1210 and P388 strains, which gave quite a short survival period of 8 approximately 9 days, RJ did not show any antitumor effect. In the case of the therapeutic RJ application employing the Sarcoma-180 ascites tumor, which gave a moderate survival period of 16 days, the increased life span was 9.3 approximately 19.3%; and with the Ehrlich ascites tumor (survival period of 22.1 days), the increased life span was 20.4% (RJ 10 mg/kg . day) and 17.6% (RJ 1,000 mg/kg . day), but no antitumor effect was observed at the dose of 100 mg/kg . day. In the case of the therapeutic experiment employing Ehrlich solid tumor, tumor growth inhibition was 25.3 approximately 54.8%, where as the use of the prophylactic-therapeutic regimen gave a tumor growth inhibition of 38.3 approximately 45.7%. In the case of the therapeutic RJ application employing Sarcoma-180 solid tumor, tumor growth inhibition was 45.1 approximately 59.7%, where as the prophylactic-therapeutic regimen gave a tumor growth inhibition of 49.1 approximately 56.1%.(ABSTRACT TRUNCATED AT 250 WORDS)