Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

Targeted dendrimer-based contrast agents for articular cartilage assessment by MR imaging.

OBJECTIVE: Magnetic resonance (MR) imaging with contrast media has shown promise for articular cartilage assessment. Dendrimer-linked nitroxides, a new family of MR contrast agents targeted to glycosaminoglycan, may improve cartilage evaluation. This study is designed to determine the ability of dendrimer-linked nitroxides to enhance articular cartilage and measure the intra-articular life-time of these agents. DESIGN: Cartilage T(1) was evaluated using immature bovine patella in solutions of five different dendrimer-linked nitroxides, saline or Gd-DTPA at 1.5T. The "relaxivity per dose" (change in cartilage 1/T(1) produced by a given concentration of agent) was calculated. The half-life of joint fluid enhancement was measured at 2T after solutions of three dendrimer-linked nitroxides, Gd-DTPA, and saline were injected into rabbit stifle joints. Twenty-four hours after injection, the joints were examined grossly and by histology for toxicity. RESULTS: All but the largest dendrimer-linked nitroxide were able to intensely enhance articular cartilage on MR. Relaxivity per dose measurements were between 3.5 and 68 times greater than Gd-DTPA. The largest nitroxide appeared to be excluded from articular cartilage. Intra-articular half-lives of the dendrimer-linked nitroxides were sufficiently long (160-208 min) for in vivo MR imaging to be performed. Histological assessments of joints showed minimal synovial inflammatory and necrosis scores 1 day post-injection that were similar for all agents, including Gd-DTPA. CONCLUSION: Dendrimer-linked nitroxides strongly enhance cartilage and are promising as articular cartilage-specific MR contrast agents. The intra-articular life-time is sufficient for imaging studies and, in initial evaluation, the agents exhibit minimal toxicity in rabbit joints.

Spin-trapping study on the hydroxyl radical formed from a tea catechin-Cu(II) system.

A spin-trapping method was applied to examine the formation of the hydroxyl (OH) radical from a tea catechin-Cu(II) system to elucidate a previous result that some tea catechin-Cu(II) systems induced DNA scission. Three tea catechins, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC), were used. The spin-trapping agent, 5,5'-dimethyl-pyrroline-1-oxide (DMPO), was dissolved in a pH 9 phosphate buffer solution, then a catechin and Cu(II) were added in that order, and the ESR spectral change was monitored for one hour. The order of adding the catechin and Cu(II) was then reversed, and the ESR spectral change was again monitored to examine the coordinating activity of each catechin toward the Cu(II) ion and the effect on OH radical generation. The intensity changes of the spin adducts, DMPO-OH, DMPO-CH3 and DMPO-H, were analyzed, the results suggesting that the OH radical generated in the system decomposed DMPO, resulting in the formation of DMPO-CH3 and DMPO-H. The results show that EGC formed a stable complex with Cu(II) and generated the OH radical. EGCg seemed to have this activity, but the OH radical that was generated was scavenged by the gallate group existing in the complex. EC did not show strong coordinating and OH-generating activities. These characteristics of the three catechins are consistent with the results shown for DNA scission.

Preoperative intradermal acupuncture reduces postoperative pain, nausea and vomiting, analgesic requirement, and sympathoadrenal responses.

BACKGROUND: In a controlled and double-blind study, the authors tested the hypothesis that preoperative insertion of intradermal needles at acupoints 2.5 cm from the spinal vertebrae (bladder meridian) provide satisfactory postoperative analgesia. METHODS: The authors enrolled patients scheduled for elective upper and lower abdominal surgery. Before anesthesia, patients undergoing each type of surgery were randomly assigned to one of two groups: acupuncture (n = 50 and n = 39 for upper and lower abdominal surgery, respectively) or control (n = 48 and n = 38 for upper and lower abdominal surgery, respectively). In the acupuncture group, intradermal needles were inserted to the left and right of bladder meridian 18-24 and 20-26 in upper and lower abdominal surgery before induction of anesthesia, respectively. Postoperative analgesia was maintained with epidural morphine and bolus doses of intravenous morphine. Consumption of intravenous morphine was recorded. Incisional pain at rest and during coughing and deep visceral pain were recorded during recovery and for 4 days thereafter on a four-point verbal rating scale. We also evaluated time-dependent changes in plasma concentrations of cortisol and catecholamines. RESULTS: Starting from the recovery room, intradermal acupuncture increased the fraction of patients with good pain relief as compared with the control (P < 0.05). Consumption of supplemental intravenous morphine was reduced 50%, and the incidence of postoperative nausea was reduced 20-30% in the acupuncture patients who had undergone either upper or lower abdominal surgery (P < 0.01). Plasma cortisol and epinephrine concentrations were reduced 30-50% in the acupuncture group during recovery and on the first postoperative day (P < 0.01). CONCLUSION: Preoperative insertion of intradermal needles reduces postoperative pain, the analgesic requirement, and opioid-related side effects after both upper and lower abdominal surgery. Acupuncture analgesia also reduces the activation of the sympathoadrenal system that normally accompanies surgery.

Induction of plant gp91 phox homolog by fungal cell wall, arachidonic acid, and salicylic acid in potato.

The oxidative burst has been suggested to be a primary event responsible for triggering the cascade of defense responses in various plant species against infection with avirulent pathogens or pathogen-derived elicitors. The molecular mechanisms of rapid production of active oxygen species (AOS), however, are not well known. We isolated homologs of gp91 phox, a plasma membrane protein of the neutrophil NADPH oxidase, from a potato cDNA library. Molecular cloning of the cDNA showed that there are two isogenes, designated StrbohA and StrbohB, respectively. The RNA gel blot analyses showed that StrbohA was constitutively expressed at a low level, whereas StrbohB was induced by hyphal wall components (HWC elicitor) from Phytophthora infestans in potato tubers. Treatment of potato tubers with HWC elicitor caused a rapid but weak transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6 to 9 h after treatment (phase II). Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocked both bursts, whereas pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolished only the second burst. These results suggest that the expression of StrbohA and StrbohB contributes to phase I and II bursts, respectively. The same is true for arachidonic acid, a lipid component of P. infestans-stimulated biphasic oxidative burst, whereas an endogenous signaling molecule, salicylic acid, only induced a weak phase II burst. Both molecules induced the StrbohB expression, which is in agreement with the second burst. To characterize the signal transduction pathway leading to the oxidative burst, we examined the role of protein phosphorylation in HWC-stimulated StrbohB gene expression. K252a and staurosporine, two protein kinase inhibitors, blocked the transcript accumulation. Two inhibitors of extracellular Ca2+ movement, however, did not abolish the transcript accumulation of StrbohB, suggesting that certain calcium-independent protein kinases are involved in the process of StrbohB gene expression. Additionally, we examined a causal relationship between the oxidative burst and expression of defense genes induced by the HWC elicitor. The transcript accumulation of genes related to sesquiterpenoid phytoalexin synthesis (lubimin and rishitin) and phenylpropanoid pathway was inhibited slightly by the DPI treatment, suggesting that the oxidative burst is not essential to activate these genes. Interestingly, the concomitant presence of DPI with the elicitor resulted in an increase in lubimin accumulation and a decrease in rishitin accumulation. Because it is known that lubimin is metabolized into rishitin via oxylubimin, we propose that AOS mediates the synthesis of rishitin from lubimin.

cDNA cloning of sesquiterpene cyclase and squalene synthase, and expression of the genes in potato tuber infected with Phytophthora infestans.

Sesquiterpene cyclase and squalene synthase are key branch point enzymes in isoprenoid pathway for the synthesis of sesquiterpenoid phytoalexins and sterols/steroid glycoalkaloids, respectively. cDNA clones encoding these enzymes were isolated from potato. A phylogenetic tree showed that the sesquiterpene cyclase is vetispiradiene synthase. Infection of Phytophthora infestans with potato tubers caused transient increases in the transcript level of vetispiradiene synthase in a compatible and an incompatible interactions. On the other hand, wound-induced expression of the squalene synthase was suppressed in favor of the expression of vetispiradiene synthase regardless of inoculated races.

A new model of white matter injury in neonatal rats with bilateral carotid artery occlusion.

Periventricular leukomalacia is an important cause of cerebral palsy and characterized by cysts and coagulation necrosis in the periventricular white matter. Since no model of periventricular leukomalacia has been established in small animals, it is expected to establish a new model of white matter injury in immature rodents. Bilateral carotid arteries were occluded in neonatal rats at 5 days of age, and the brain neuropathologically examined at 7 days of age. Among 22 brains histologically examined, 20 (90.9%) had white matter changes including coagulation necrosis and cystic lesions in and around the internal capsule, while only two had small cerebral infarction and five showed some ischemic neurons in the cerebral cortex. Cerebral blood flow (CBF) decreased to about 25% of controls in the subcortical white matter in the animals with bilateral carotid artery occlusion (BCAO). Amyloid precursor protein (APP) immunohistochemistry demonstrated various APP-immunoreactive axonal profiles in the internal capsule and the subcortical white matter, and stronger expression of APP in pyramidal neurons in the cerebral cortex of BCAO brains. These results indicated that the white matter is more vulnerable than the cerebral cortex in 5-day-old rats when CBF decreases to about 25% and suggested that this model is useful for investigating the white matter changes induced by cerebral hypoperfusion in the neonatal brain, since previous models of hypoxic-ischemic brain injury in neonatal mice and rats revealed preferential susceptibility of the gray matter. It was also indicated that APP is a sensitive marker for mild axonal disruption in the white matter of the immature brain.

Total body bone development during early childhood in very low birth weight infants without cerebral palsy and mental retardation.

Total body bone mineral density was measured by dual energy X-ray absorptiometry in 52 children who were very low birth weight (VLBW) infants without cerebral palsy and mental retardation (postconceptional age, from 10 mo to 6 y and 6 mo). VLBW infants in this study seemed to show compensatory acceleration of total body bone development, catching up with the control group during early childhood. However, in VLBW infants with at least one of the three factors such as total parenteral nutrition for 1 week or more, assisted ventilation for 1 week or more, or oxygen therapy for 28 d or more in their early stage after birth, adequate mineral supplementation might be especially important for long-term bone development.

Beta ray-induced scission of DNA in tritiated water and protection by a green tea percolate and (-)-epigallocatechin gallate.

The beta-ray induced scission of puC18 plasmid DNA from E. coli in tritiated water was examined in the presence or absence of a green tea percolate (TP) and the main constituent, (-)-epigallocatechin gallate (EGCg). An analysis of the ratio of the original closed-circular to the open-circular form of DNA, which was formed by the strand scission of DNA, revealed that TP and EGCg showed a protective effect on DNA scission depending on their concentrations. A new technique, named solid state spin trapping, was applied to examine this scavenging ability toward the hydroxyl (OH) radical generated in tritiated water. The result was kinetically analyzed to reveal that TP and EGCg showed the scavenging effect, suggesting that the protective effect on DNA scission was attributable to the scavenging effect on the OH radical.

The oxidative burst protects plants against pathogen attack: mechanism and role as an emergency signal for plant bio-defence--a review.

Various aspects, mechanisms and functions of the oxidative burst with generation of O2- superoxide anions in plant cells, which is stimulated by active defence-inducing agents such as fungal infection or elicitor treatment, were reviewed mainly on the basis of experimental evidence obtained in a system of Solanaceae plants and Phytophthora spp. The oxidative burst may be due to an O(2-)generating NADPH oxidase in the plasma membrane, which is activated with combinations of cytosolic proteins, Ca2+, calmodulin and protein kinase, following stimulation by elicitor molecules. The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defence.

A cytosolic phospholipase A2 from potato tissues appears to be patatin.

Phospholipase (PL) A2 is involved in signal transduction in the resistance reaction that is induced in potato by inoculation of an incompatible race of Phytophthora infestans, the late blight fungus, or by treatment with fungal elicitor hyphal wall components (Kawakita et al. 1993). In this study, PLA2 in the soluble fraction from potato tuber was purified. The following results suggested that the enzyme was, in fact, patatin: (1) the molecular mass of the purified enzyme was 40 kDa, the same as that of patatin; (2) the pI of the purified enzyme was approximately 4.75, which corresponds to that of patatin; and (3) the amino-terminal amino acid sequence of the purified enzyme showed a high degree of homology to that of patatin. Patatin is known as a storage protein of the potato tuber and it has been shown to have esterase activity. However, other enzymatic activities and the function(s) of patatin are unknown. We investigated the PLA activities of the purified patatin. The PLA2 activity of the patatin was much higher than the PLA1 activity, even though the protein exhibited both activities. The PLA2 activity of the enzyme was particularly apparent when phosphatidylcholine with linoleic acid at the sn-2 position was used as substrate. Lower activity was observed with phosphatidylcholine with palmitic acid, oleic acid and arachidonic acid at the sn-2 position.

Protecting effect of a green tea percolate and its main constituents against gamma ray-induced scission of DNA.

Gamma ray-induced scission of puC18 plasmid DNA prepared from E. coli was examined in the presence of a green tea percolate and its main constituents, L-ascorbic acid (used as the sodium salt) and (-)-epigallocatechin gallate. Each of these showed a protecting effect against DNA scission. The relationship between the protecting effect against DNA scission and the scavenging effect of the hydroxyl radical was examined, and is discussed from the viewpoint of interaction with DNA.

Effects of ACTH treatment on cerebral phosphorus metabolites in infants with the West syndrome.

Concentrations of phosphorus metabolites in brains of 4 infants with the West syndrome were measured by 31P MRS before and during ACTH treatment. Although intracellular pH, phosphocreatine/ATP ratio and phosphocreatine/inorganic phosphate ratio in the patients were similar to those in the controls and did not change after the ACTH treatment, phosphomonoesters/phosphodiesters ratio in the patients always decreased with ACTH treatment. These results indicate that ACTH may accelerate the maturation of phospholipids metabolism in infant brains, and the observed change may relate to the efficacy of ACTH treatment against the West syndrome.

A case of pancreatic cancer with the sign of Leser-Trélat.

A case of 79-year-old man with pancreatic cancer associated with the sign of Leser-Trélat was presented. Abdominal CT scanning was performed and a pancreatic tail tumor involving the spleen and the greater omentum as well as ascitic retention were detected. Ba-enema study showed the stenosis of the sigmoid colon, suggesting the peritoneal disseminations. Absolute non-curative operation of side-to-side transverso-sigmoidostomy was performed for colonic stenosis, and biopsy of the disseminated omental tumor was performed, which was histologically diagnosed as well differentiated papillary adenocarcinoma compatible with pancreatic cancer. The postoperative course was uneventful and at present the patient was followed at outpatient clinic. To our knowledge, this case is the first report with pancreatic cancer associated with the sign of Leser-Trélat in Japan.

P-31 MR spectrum and histologic changes after intrahepatic arterial injection of iodized oil in normal and cirrhotic rat liver.

Injection of iodized oil (Lipiodol) into the hepatic artery is widely used in the diagnosis and treatment of hepatocellular carcinoma. However, no reports have yet appeared concerning temporal changes in hepatic metabolism following Lipiodol injection. In the present study, Lipiodol was injected into the hepatic arteries of normal and cirrhotic rats, successive P-31 MR measurements were performed, and temporal changes in metabolism were compared with histologic findings. Both normal and cirrhotic rats displayed minimum levels of beta-ATP/PME and beta-ATP/Pi 5 days after hepatic arterial injection of Lipiodol. However, 10 days after injection these values had reverted to the preinjection levels. The metabolic dysfunction observed in the liver following hepatic arterial injection of 0.3 ml/kg b.w. Lipiodol was transient. Moreover, no distinct differences were observed between P-31 MR changes in normal and cirrhotic rats. Conversely, histologic impairment assessed on the basis of hepatic necrosis ratios was most severe 2 days after hepatic arterial injection in both normal and cirrhotic rats, and this did not coincide with the time of the most pronounced metabolic impairment as inferred from P-31 MR changes.

Effectiveness of Lipiodol in transcatheter arterial embolization of hepatocellular carcinoma.

The effectiveness of Lipiodol (iodized oil) in transcatheter arterial embolization (TAE) of hepatocellular carcinoma (HCC) was retrospectively evaluated using statistical analysis. A total of 343 HCC patients who underwent TAE at 5 institutions between 1984 and 1989 were divided into 2 groups: the GS-TAE group underwent TAE with Gelfoam sponge alone, whereas the LP-TAE group was given Lipiodol (LP) immediately before GS-TAE. The statistical T value calculated for the LP-TAE group showed that the administration of LP, the tumor size, intrahepatic metastasis, portal vein infiltration, and serum total bilirubin and alpha-fetoprotein levels significantly (P < 0.01) affected the patients' survival. Both the cumulative survival determined using the Kaplan-Meier model and the cumulative hazard calculated using Cox's proportional hazard model differed significantly (P < 0.01) between the GS-TAE group and the LP-TAE group (log-rank test). These results confirmed the effectiveness of LP used in combination with Gelfoam sponge for TAE of HCC.

Adipocyte dynamics in hypothalamic obese mice during food deprivation and refeeding.

Effect of hypothalamic lesions on regulation of body weight and fat cell dynamics in obese mice were examined during refeeding after prolonged food deprivation. Obese mice, which were treated with monosodium glutamate for 5 postnatal days and had ventromedial nuclear lesions in the hypothalamus, were used. When adult obese mice were given a glucose electrolyte solution for 20-40 days, the body weight dropped to about 45% of their pre-treatment weight. After reinstituted feeding of normal mouse food ad libitum, their body weight and adipose tissue weight returned to pre-starvation level. Tritiated thymidine autoradiography revealed that cell proliferation occurred in the early stages of refeeding and some fat cells were renewed in the epididymal adipose tissue. Fat cell renewal was found more active in the experimental group than in the control. Thereafter, fat cell size increased gradually via fat storage. These obese mice were found to have the capacity to regulate their body weight and adipose tissue not only through fat storage but also by increasing number of fat cells, in order to replace the cells which were lost during starvation. Therefore, ventromedial nuclear lesion in the hypothalamus does not influence the regulatory mechanism of adipose tissue during starvation and refeeding.

[Effect of intraarterially injected lipiodol on rat liver with cirrhosis].

The serial histological changes of the liver following the intrahepatic arterial injection of Lipiodol in the rat with cirrhosis were investigated. The hepatocytes showed acidophilic degeneration and focal necrosis after 12 hours and restoration of focal necrosis was seen after 72 hours. Necrosis and infarction were resolved after 120 hours. We concluded that the intrahepatic arterial injection of 0.1 ml Lipiodol in the rat with cirrhosis was safely performed.

The influence of hyperthermia in vitro on the functions of peritoneal macrophages in mice.

Total-body hyperthermia (TBHT) as a treatment for cancer may lead to a reduction in the host's immunocompetence as a result of the direct effects of heat on the immune system. Thus, we studied the influences of hyperthermia in vitro on the function of peritoneal macrophages from mice. Peritoneal macrophages from C3H/HeN mice were heated in vitro for 3 hr at 37, 39, 40, 41 or 42 degrees C. After exposure to heat, the phagocytic ability of the macrophages, as well as results of the nitroblue tetrazolium (NBT) reduction test and the cytotoxicity test were examined. The changes in all these parameters showed almost the same pattern: a tendency for macrophage functions to be potentiated up to 40 degrees C, and a tendency towards inhibited functioning at temperatures above 41 degrees C. Although augmented functions of macrophages were observed after exposure to mild hyperthermia (less than 40 degrees C), the possibility of TBHT (42 degrees C)-induced inhibition of macrophage function must be further investigated in clinical trials of TBHT therapy for cancer.

MR imaging of the liver before and after transcatheter hepatic chemo-embolization for hepatocellular carcinoma.

Transcatheter chemo-embolization (TCE) in hepatocellular carcinoma (HCC) was performed in 38 patients. The patients were examined by MR imaging before TCE as well as one week and 4 to 5 weeks after TCE. The tumor signal intensity in T2 weighted images increased in 13 cases and decreased in 19 cases after TCE. Increased intensity seemed to reflect intra-tumoral hemorrhage or liquefaction accompanying tumor necrosis. Decreased intensity seemed to reflect coagulation necrosis. In 9 of 18 cases followed over a 2-month period the signal intensity had decreased in both T1 and T2 weighted images. In these patients the tumor showed no recurrence at angiography and the decreased signal seemed to reflect the completion of coagulation necrosis. A hyper- and/or hypointense rim around the tumor appeared in 22 cases. These changes were thought to be perifocal edema or granulation tissue around the tumor. MR imaging was useful in evaluating the necrotic process of the tumor after TCE.