Chronic social defeat stress increases dopamine D2 receptor dimerization in the prefrontal cortex of adult mice.

The present study aimed to examine the effects of chronic social defeat stress on the dopamine receptors and proteins involved in post-endocytic trafficking pathways. Adult mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. Western blot analysis was used to measure the protein expression levels of dopamine D2 receptors (D2Rs), a short (D2S) and a long form (D2L) and, D2R monomers and dimers, dopamine D1 receptors (D1Rs), neuronal calcium sensor-1 (NCS-1) and G protein-coupled receptor-associated sorting protein-1 (GASP-1), and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression levels of D2S, D2L, D2R monomers and dimers, and D1Rs in different brain areas. We observed increased expression of D2S, D2L and D2Rs dimers in the prefrontal cortex (PFC) of susceptible and/or unsusceptible mice compared with controls. The only significant findings with regard to mRNA expression levels were lower expression of D2S mRNA in the amygdala (AMYG) of susceptible and unsusceptible mice compared with controls. The present study demonstrated that chronic social defeat stress induced increased expression of D2S, D2L, and D2R dimers in the PFC of susceptible and/or unsusceptible mice.

ER Stress and Autophagy.

Eukaryotic cells respond to various types of stresses caused by changes in the extracellular environment. Intracellular factors, such as the accumulation of misfolded proteins in the endoplasmic reticulum (ER), also cause stress and activate the unfolded protein response (UPR), which induces the expression of chaperones and proteins involved in the recovery process. However, if the stress is excessive or sustained, and ER function cannot be restored, the UPR triggers apoptosis, thereby removing the affected cell. It is now apparent that ER stress is also a potent trigger for autophagy, a self-degradative process that has an adaptive function. This review surveys the intersection of ER stress and autophagy and highlights the potential therapeutic implications thereof.

The characteristics of Bax inhibitor-1 and its related diseases.

Bax inhibitor-1 (BI-1) is an evolutionarily-conserved endoplasmic reticulum protein. The expression of BI-1 in mammalian cells suppresses apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. BI-1 has been shown to be associated with calcium (Ca(2+)) levels, reactive oxygen species (ROS) production, cytosolic acidification, and autophagy as well as endoplasmic reticulum stress signaling pathways. According to both in vitro and clinical studies, BI-1 promotes the characteristics of cancers. In other diseases, BI-1 has also been shown to regulate insulin resistance, adipocyte differentiation, hepatic dysfunction and depression. However, the roles of BI-1 in these disease conditions are not fully consistent among studies. Until now, the molecular mechanisms of BI-1 have not directly explained with regard to how these conditions can be regulated. Therefore, this review investigates the physiological role of BI-1 through molecular mechanism studies and its application in various diseases.

BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis.

Endoplasmic reticulum (ER) stress is considered one of the pathological mechanisms of idiopathic pulmonary fibrosis (IPF). Therefore, we examined whether an ER stress regulator, Bax inhibitor-1 (BI-1), regulates collagen accumulation, which is both a marker of fibrosis and a pathological mechanism of fibrosis. The presence of BI-1 inhibited the transforming growth factor-ß1-induced epithelial-mesenchymal transition of epithelial pulmonary cells and bleomycin-induced pulmonary fibrosis in a mouse model by enhancing collagen degradation, most likely by enhanced activation of the lysosomal V-ATPase through glycosylation. We also found a correlation between post-translational glycosylation of the V-ATPase and its associated chaperone, calnexin, in BI-1-overexpressing cells. BI-1-induced degradation of collagen through lysosomal V-ATPase glycosylation and the involvement of calnexin were confirmed in a bleomycin-induced fibrosis mouse model. These results highlight the regulatory role of BI-1 in IPF and reveal for the first time the role of lysosomal V-ATPase glycosylation in IPF.

Microbiological and clinical characteristics of bacteraemia caused by the hypermucoviscosity phenotype of Klebsiella pneumoniae in Korea.

Hypermucoviscous (HV) isolates of Klebsiella pneumoniae have been linked to virulence potential in experimental infections. We examined 33 isolates of K. pneumoniae from patients with bacteraemia for the HV phenotype on agar culture, and determined their virulence potential by screening for capsular (K) serotype by polymerase chain reaction and the presence of seven virulence factor genes. Fourteen (42·4%) isolates expressed the HV phenotype and 11 of these were serotype K1 or K2; these serotypes were not identified in HV-negative isolates. The genes rmpA, rmpA2, aerobactin, wabG and allS were significantly more frequent in HV than non-HV isolates. Multilocus sequence typing identified 21 sequence types (ST), eight of which were found in HV-positive isolates and the clonal relatedness of isolates of the most frequent types (ST23 and ST11) from different hospitals was confirmed by pulsed-field gel electrophoresis. The HV phenotype was more associated with community-acquired infection with a lower frequency of fatal underlying illness, but with significantly more focal infections, notably liver abscesses. Clinicians should be aware of such clinical impacts of the HV phenotype.

Safety of protected carotid artery stenting in patients with severe carotid artery stenosis and carotid intraplaque hemorrhage.

BACKGROUND AND PURPOSE: Carotid IPH can be detected with MR imaging. The aim of this study was to determine the safety of CAS using an emboli protection device in patients with severe carotid artery stenosis and MR imaging-depicted carotid IPH. MATERIALS AND METHODS: We retrospectively reviewed a prospective data base that included 91 consecutive patients with severe carotid stenosis and high-risk features who were treated with CAS by using an emboli protection device. Seventy-eight of the included patients underwent prestenting 3D TOF MRA. IPH was defined as the presence of high signal intensity within the carotid plaque, greater than 150% of the signal intensity of the adjacent neck muscle on TOF source images. The primary outcome measure was the combined incidence of stroke, MI, and death within 30 days of CAS. Associations between IPH and the primary outcome were investigated. RESULTS: IPH was detected on TOF MRA in 30 patients. Symptomatic patients were more common in the IPH group than in the non-IPH group (66.7% vs 41.7%; P = .032). Overall, 30-day stroke, MI, or death rates were 6.6%. There was no significant difference in the primary outcome between the IPH and non-IPH groups (10% and 6.25%, respectively; hazard ratio for IPH, 1.151; 95% CI, 0.035 to 37.500; P = .937). A logistic regression showed there was no independent variable associated with the primary outcome. CONCLUSIONS: The results of this study indicate that protected CAS seems to be safe in patients with severe carotid stenosis and IPH.

Immature Rubus coreanus Shows a Free Radical-Scavenging Effect and Inhibits Cholesterol Synthesis and Secretion in Liver Cells.

Rubus coreanus fruits have been employed as a traditional medicine for centuries in the Asia-Pacific region. Its pharmacological action differs according to the different extraction methods utilized and the degree of fruit ripening. In this study, we determined the cellular effect of different ethanol extracts of mature and immature Rubus coreanus fruits in human hepatic cell line, HepG2 cells. The antioxidant activity, effect on superoxide dismutase activity and cholesterol biosynthesis efficiency was also evaluated. Immature Rubus coreanus extract showed higher antioxidant capability, compared with that of its mature fractions. Cellular antioxidant proteins including HO-1, Cu/Zn-superoxide dismutase and catalase were highly expressed in the presence of Rubus coreanus. Cholesterol levels in HepG2 cells treated with the water fraction of immature Rubus coreanus were significantly reduced. This antihyperlipidaemic action of Rubus coreanus is a consequence of cholesterol biosynthesis and extracellular secretion in HepG2 cells. These results indicate that among different ethanol fraction of mature and immature Rubus coreanus fruit extracts, water extract of immature fruit extract shows higher antioxidant as well as higher antihyperlipidaemic action.

BAX inhibitor-1 enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger: the alteration of mitochondrial function.

The anti-apoptotic protein, BAX inhibitor-1 (BI-1), has a role in cancer/tumor progression. BI-1-overexpressing HT1080 and B16F10 cells produced higher lung weights and tumor volumes after injection into the tail veins of mice. Transfection of BI-1 siRNA into cells before injection blocked lung metastasis. in vitro, the overexpression of BI-1 increased cell mobility and invasiveness, with highly increased glucose consumption and cytosolic accumulation of lactate and pyruvate, but decreased mitochondrial O(2) consumption and ATP production. Glucose metabolism-associated extracellular pH also decreased as cells excreted more H(+), and sodium hydrogen exchanger (NHE) activity increased, probably as a homeostatic mechanism for intracellular pH. These alterations activated MMP 2/9 and cell mobility and invasiveness, which were reversed by the NHE inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting a role for NHE in cancer metastasis. In both in vitro and in vivo experiments, C-terminal deleted (CDeltaBI-1) cells showed similar results to control cells, suggesting that the C-terminal motif is required for BI-1-associated alterations of glucose metabolism, NHE activation and cancer metastasis. These findings strongly suggest that BI-1 reduces extracellular pH and regulates metastasis by altering glucose metabolism and activating NHE, with the C-terminal tail having a pivotal role in these processes.

Enzymatic oxidation of aqueous pentachlorophenol.

The influence of the nonionic surfactant Tween 80 on pentachlorophenol (PCP) oxidation catalyzed by horseradish peroxidase was studied. The surfactant was tested at concentrations below and above its critical micelle concentration (CMC). Enhancement of PCP removal was observed at sub-CMCs. The presence of Tween 80 in the reaction mixture reduced enzyme inactivation which occurred through a combination of free radical attack and sorption by precipitated products. A simple first-order model was able to simulate time profiles for enzyme inactivation in the presence or absence of Tween 80. At supra-CMCs, the surfactant caused noticeable reductions in PCP removal, presumably through micelle partitioning of PCP which precluded the hydrophobic PCP molecule from interacting with the enzyme.

Characterization of cytochrome P450s mediating ipriflavone metabolism in human liver microsomes.

Ipriflavone, a synthetic flavonoid for the prevention and treatment of osteoporosis, has been reported to be extensively metabolized in man to seven metabolites (M1-M7). This study was performed to characterize the human liver cytochrome P450s (CYP) responsible for the metabolism of ipriflavone. Hydroxylation at the beta-ring to M3, O-dealkylation to M1 and oxidation at isopropyl group to M4 and M5 are major pathways for ipriflavone metabolism in three different human liver microsome preparations. The specific CYPs responsible for ipriflavone oxidation to the active metabolites, M1, M3, M4 and M5 were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potencies of ipriflavone and its five metabolites, M1-M5 on seven clinically important CYPs were investigated in human liver microsomes. Our results demonstrate that CYP3A4 plays the major role in O-dealkylation of ipriflavone to M1 and CYP1A2 plays a dominant role in the formation of M3, M4 and M5. Ipriflavone and/or its five metabolites were found to inhibit potently the metabolism of CYPs 1A2, 2C8, 2C9 and 2C19 substrates.

Placenta hominis protects osteoporosis in ovariectomized rats.

In China, Japan, and Korea, placenta hominis extracts (PHEs) are used clinically for the treatment of osteoporosis. The anti-osteoporotic effect of PHEs was studied. The trabecular bone area and thickness in OVX rats decreased by 50% from those in sham-operated rats; these decreases were completely inhibited by administration of PHEs for 7 weeks. Osteoclast numbers and the osteoblast surface were enhanced in OVX rats, but PHEs had no effect on these phenomena. Serum phosphorus and alkaline phosphatase in OVX rats increased compared to those in sham-operated rats, but the increases were not affected by the administration of PHEs. Thyroxine (T4) level was stimulated in OVX rats. The extracts inhibited the T4 level in the OVX rats. These results strongly suggest that PHEs be effective in preventing the development of bone loss induced by OVX in rats.

Mechanism of cyclosporine-induced overgrowth in gingiva.

UNLABELLED: Cyclosporine A (CsA) is a widely used immunosuppressant but with significant side-effects, such as gingival overgrowth. This study investigates how CsA induces gingival proliferation and shows the effects of the CsA-associated signaling messengers, IL-6 and TGF-beta1, on gingival proliferation. CsA increased both IL-6 and TGF-beta1 levels. In addition to CsA, an IL-6 or TGF-beta1 treatment also induced gingival fibroblast proliferation. Inhibiting the cytokine resulted in the suppression of CsA-induced proliferation. MAPKs and PI3K are known to be involved in cell proliferation. Therefore, the effect of CsA on the kinase activities was examined. The results showed that both p38 MAPK and PI3K are essential for gingival fibroblast proliferation. TGF-beta1 and IL-6 and their associated signaling transduction may be novel bona fide molecular targets for the prevention of gingival overgrowth in CsA-treated patients. ( ABBREVIATIONS: MAPK, mitogen-activated protein kinase; P13K, phosphatidylinositol 3-kinase.)

Carbon monoxide and nitric oxide protect against tumor necrosis factor-alpha-induced apoptosis in osteoblasts: HO-1 is necessary to mediate the protection.

BACKGROUND: Carbon monoxide (CO) and nitric oxide (NO) each have unique roles for various inflammatory states, including inflammatory bone resorption. Although it is known that NO can induce the expression of the cytoprotective enzyme HO-1, there is no information as to whether the protective effect of CO requires NO production or whether CO must induce the expression of HO-1 to exert its functional effects. METHODS: Murine osteoblast cells, MC3T3E1 osteoblasts, were cultured for CO and NO-associated HO-1 experiments and were transfected with pcDNA 3, pcDNA 3-HO-1, control siRNA or HO-1 siRNA using Nucleofector. For cell death measurement, MTT and annexin V assays were used. We performed Western blotting to check the expressions of HO-1 and iNOs and measured the HO-1 enzyme activity. We also measured the amounts of nitrite and nitrate using Griess reagents. RESULTS: The increased expression of HO-1 is required for the protective effect of NO and a single treatment of CO can increase the expression of HO-1, and this is also important for the protective effect of CO in MC3T3E1 osteoblasts. CO as well as NO attenuates the TNF-alpha-induced apoptosis in osteoblasts. The anti-apoptotic effect of CO or NO is not mediated by cGMP, and CO has no effect on the release of NO. The inhibition of HO-1 with using the HO-1 inhibitor ZnPP or HO-1 siRNA resulted in a striking increase of apoptosis in the CO/TNF-alpha-treated cells. Furthermore, HO-1 overexpression showed resistance against the TNF-alpha-induced cytotoxicity in the MC3T3E1 osteoblasts. CONCLUSIONS: There is a need for HO-1 expression to mediate the protection provided by exogenous CO or NO in osteoblasts.

p38 MAPK and NF-kappaB on IL-6 release in human gingival fibroblasts.

The induction of interleukin-6 (IL-6) using a proinflammatory cytokine (IL-1beta) was studied in human gingival fibroblasts (HGFs) in relation to p38 MAPK and NF-kappaB transcription factor. When added to HGFs, IL-1beta had a stimulatory effect on the production of IL-6, and this effect was significantly reduced by SB203580, a specific p38 MAPK inhibitor. In addition, the stimulation of IL-6 release also was reduced by the addition of pyrrolidine dithiocarbamate or NF-kappaB SN50, which has been reported as potent NF-kappaB inhibitor. Both the NF-kappaB inhibitors in the presence of SB203580 had more inhibitory effect on IL-6 release. IL-13 stimulated NF-kappaB binding affinity as well as p38 MAP kinase activation, leading to the release of IL-6. However, a specific inhibitor of p38 MAPK, SB203580, had no effect on the NF-kappaB activation, and both the NF-kappaB inhibitors failed to reduce the p38 MAPK activation in the IL-1beta-stimulated HGFs. These results strongly suggest that both p38 MAPK and NF-kappaB are required in IL-1beta-induced IL-6 synthesis and that these two IL-1beta-activated pathways can be primarily dissociated.

Prevention of bone loss in ovariectomized rats: the effect of Salvia miltiorrhiza extracts.

The preventive effect of Salvia miltiorrhiza extracts (SMEs) on the progress of bone loss induced by ovariectomy (OVX) was studied in rats. We measured body weight and bone histomorphometry in sham, OVX or SMEs-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham rats and in SMEs-administered rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of SMEs for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of SMEs for 7 weeks. In OVX rats, free T3 was normal in all cases, whereas free T4 was significantly increased. Although there was no difference between OVX and SMEs-administered rats in T3 level, we have found significant difference between them in T4 level. These results strongly suggest that SMEs are effective in preventing the development of bone loss induced by OVX in rats.

Yuk-Hap-Tang induces apoptosis by intervening mn-SOD in human cervical carcinoma HeLa cells.

Yuk-Hap-Tang (YHT) induces cell death in human cervical carcinoma HeLa cells. Caspase-3, -6 and -9 were markedly activated in HeLa cells treated with YHT. The preferred substrate for caspase-3 cysteine protease, PARP, was cleaved to its 85-kDa cleavage product. YHT increased the amount of the anti-apoptotic protein, Bcl-2, and the pro-apoptotic protein, Bax. Although p53 has been reported to accumulate in cancer cells in response to anticancer agents, the p53 expression level was not changed in HeLa cells treated with YHT. Manganese (Mn)-TBAP, a mitochondria-specific SOD mimetic agent and NAC/GSH (N-acetyl cysteine/ reduced glutathione) reduced the YHT-induced cytotoxicity and decreased the number of the YHT-induced apoptotic cells. Furthermore, YHT reduced the expression of Mn-SOD protein and its activity in HeLa cells. The data demonstrate that YHT induces the apoptosis of human cervical carcinoma HeLa cells by intervening Mn-SOD.

ERK MAP Kinase is required in 1,25(OH)2D3-induced differentiation in human osteoblasts.

Expression of alkaline phosphatase(ALP)activity represents a key event during the differentiation processes of osteoblasts, and the level of ALP activity has been routinely used as a relative measure of differentiation stages of osteoblasts. In human osteoblasts, we showed that vitamin D3 analogue, 1,25(OH)2D3, had a stimulatory effect on ALP activity after 3 days, compared with control. The treatment of PD098059, an ERK MAP Kinase inhibitor, had a reducing effect on ALP activity, a differentiation marker in 1,25(OH)2D3-treated primary human osteoblasts. However, SB203580, a potent p38 MAP Kinase inhibitor, had no effect on the differentiation in this system. This indicates that ERK, not p38, is directly related to 1,25(OH)2D3-stimulated ALP activity in primary human osteoblasts. These results also show that the vitamin D3 analogue stimulates ERK1 activation in primary human osteoblasts. This finding provides one of signaling pathways for differentiation in primary human osteoblasts.

Inhibition of tumour necrosis factor-alpha secretion from rat astrocytes by Sesim-Tang.

Substance P (SP) can stimulate secretion of tumour necrosis factor-alpha (TNF-alpha) from astrocytes stimulated with lipopolysaccharide (LPS). In this study, we have examined whether an aqueous extract of Sesim-Tang inhibits the secretion of TNF-alpha from primary cultures of rat astrocytes. Sesim-Tang (10-1000 microg/mL) significantly inhibited the TNF-alpha secretion by astrocytes stimulated with LPS and SP. Interleukin-1 (IL-1) has been shown to elevate TNF-alpha secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore examined whether IL-1 mediated inhibition of TNF-alpha secretion from primary astrocytes by Sesim-Tang. Treatment with Sesim-Tang (10-1000 microg/mL) of astrocytes stimulated with both LPS and SP decreased IL-1 secretion significantly. Moreover, the secretion of TNF-alpha by LPS and SP in astrocytes was progressively inhibited with increasing amounts of IL-1 neutralizing antibody. Our results suggest that Sesim-Tang may inhibit TNF-alpha secretion by inhibiting IL-1 secretion and that Sesim-Tang has an antiinflammatory activity in the central nervous system.

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis.

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0-3.8 x 10(5) Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index = 1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0-5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22 degrees C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.