Reelin is a very large secreted glycoprotein that is essential for brain formation and function, but the mechanism by which it affects the dynamics and morphology of neuronal cells remains unsolved. One previous study claimed that Reelin has a proteolytic activity against extracellular matrix proteins, which might explain many of the actions of Reelin. Therefore, in this study wild-type Reelin protein and its mutant in which a supposedly critical serine residue was replaced were expressed and tested for their self-degrading and laminin-degrading activities. We found that both of these proteins generated totally the same cleaved fragments and that neither of them is capable of degrading laminin. It is thus likely that Reelin is not a serine protease and is unable to degrade extracellular matrix.

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Overexpression/amplification of human epidermal growth factor receptor (HER)2/neu (erbB-2) oncogene plays a causal role in carcinogenesis and correlates with a poor clinical prognosis. However, little is known about HER2 in gastric cancer. In this study, we explored the pharmacological activities of natural triterpenoid corosolic acid (CRA) in HER2 signaling and its role in gastric cancer development and progression. In this study, CRA dramatically inhibited HER2 expression in a dose- and time-dependent manner, effectively inhibited cell proliferation, and induced G₀/G₁ arrest through the induction of p27^kip1 and cyclin D₁ down-regulation. CRA exposure enhanced apoptotic cell death, as confirmed by caspase-3 and poly (ADP-ribose) polymerase cleavage activities. CRA inhibited signaling pathways downstream of HER2, including phospho-proteins such as Akt and Erk. In addition, CRA combined with adriamycin and 5-fluorouracil enhanced this growth inhibition, but not with docetaxel and paclitaxel. These findings demonstrate that CRA suppresses HER2 expression, which in turn promotes cell cycle arrest and apoptotic cell death of gastric cancer cells, providing a rationale for future clinical trials of CRA in the treatment of HER2-positive gastric cancers.

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Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs), which are composed of core proteins and the glycosaminoglycans that bind to them, are one of the main components of the extracellular matrix around pathogens such as bacteria. This study investigated the effects of PG extracted from the nasal septum cartilage of whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. iDCs were prepared from human monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The iDCs were stimulated by W-PG alone. In another type of experiment, the iDCs were stimulated by MIX (tumor necrosis factor-α (TNF-α), IL-1β, IL-6 and prostaglandin E₂ (PGE₂)) or a combination of MIX plus W-PG. The stimulation of W-PG alone did not induce the phenotypic maturation from iDCs. However, W-PG promoted the up-regulation of chemokine receptor CCR7-surface expression and the chemotactic responsiveness to CCR7 ligand macrophage inflammatory protein-3β on MIX-stimulated mDCs although W-PG did not influence matrix metalloproteinase-9 activity which is an important factor in DC migration through the extracellular matrix. The findings that W-PG can selectively regulate the chemotactic activity of DCs in vitro under inflammatory conditions therefore indicate that the interaction of PGs with immune cells including DCs plays an important role in the immune response under the milieu of innate immunity.

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Compound K (CK; 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol), an active ginseng saponin metabolite, exerts anticancer activity via apoptosis induction in various cancers. In the present study, we investigated the anti-angiogenic activity of CK and its molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced in HUVECS by basic fibroblast growth factor (bFGF), a potent angiogenic growth factor. CK significantly inhibited the proliferation and also attenuated the expression of a proliferating protein cyclin D1 in bFGF treated HUVECs. Also, CK significantly inhibited the migration and tube formation of bFGF treated HUVECs at non-cytotoxic concentrations, reduced secreted level of vascular endothelial growth factor (VEGF) and increased the secreted level of pigment epithelium-derived factor (PEDF) in HUVECs. In addition, CK effectively disrupted bFGF-induced neo-vascularization in the Matrigel plugs excised from mice in vivo. Notably, we have found that CK downregulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and AKT in bFGF treated HUVECs. Taken together, our findings for the first time indicate that CK exerts anti-angiogenic activity via inhibition of p38 MAPK and AKT in HUVECs with the potential of a cancer chemopreventive agent.

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Nedd4-interacting protein 2 (NDFIP2) has three transmembrane domains and interacts with multiple Nedd4 family ubiquitin ligases through polyprolinetyrosine (PY) motifs located in its N-terminal cytoplasmic domain. It has been postulated that NDFIP2 acts as an adaptor for the ubiquitylation of substrates with Nedd4 ubiquitin ligase. However, whether NDFIP2 promotes or inhibits the ubiquitylation of Nedd4 substrates is still under debate. We show here that although NDFIP2 is detected in the Golgi/trans-Golgi network (TGN) area, it is rapidly delivered to and degraded in lysosomes with its half-life ca. 1.5 h. Intriguingly, knockdown (KD) of NDFIP2 with small interfering RNA (siRNA) impaired both the formation and function of gap junctions. Indeed, KD of NDFIP2 destabilized the gap junction protein connexin43 that contains PY motif. In support of this, overexpression of NDFIP2 stabilized connexin43 and enhanced the formation of gap junctions. Furthermore, the PY motifs of NDFIP2, which are required for its interaction with Nedd4, Atrophin-1 interacting protein (AIP) 4 (AIP4)/Itch, and AIP2/WWP2, were necessary for the targeting of NDFIP2 to lysosomes and/or the stability of connexin43 and gap junctions. Collectively these findings suggest that NDFIP2 may inhibit the Nedd4-dependent ubiquitylation of membrane proteins containing PY motifs, such as connexin43, in a competitive manner.

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The aim of this study was to evaluate the prokinetic and gastric-relaxing effects of the isoquinoline alkaloid corydaline, which was extracted from Corydalis tubers (CT). Corydaline is a marker compound used for quality control of DA-9701, a prokinetic agent formulated from extracts of Pharbitidis semen and Corydalis tuber that is currently in clinical trials in Korea for the treatment of functional dyspepsia (FD). DA-9701 was previously reported to be a potential therapeutic agent for the treatment of abnormalities in gastrointestinal motor function in FD patients; however, the therapeutic effects of corydaline on FD have yet to be demonstrated in an in vivo study. In the current study, oral administration of corydaline not only significantly accelerated gastric emptying in normal rats but also improved delayed gastric emptying to near normal levels. Furthermore, corydaline induced significant gastric relaxation, shifting the pressure–volume curve towards higher volumes compared to controls. These results suggest that corydaline promotes gastric emptying and small intestinal transit and facilitates gastric accommodation.

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In an acute treatment experiment, metformin (150, 300 mg/kg, per os (p.o.)) markedly reduced the consumption of a high-fat diet (HFD) (45 kcal% fat-containing diet) for 2 h after the HFD was given to the fasted male C57BL/6J (B6) mice. In addition, metformin at a higher dose increased plasma active glucagon-like peptide-1 (GLP-1) levels at 1 h after the HFD was given. On the other hand, pioglitazone (12 mg/kg, p.o.) slightly increased the food intake but did not affect active GLP-1 levels when given at 6 and 12 mg/kg, p.o. In a long-team experiment for 9 weeks, metformin treatment (0.25, 0.5% in the HFD) resulted in reduction of body weight gain and HFD intake. When wet weights of various body fat pads of each mouse were measured at 9 weeks after treatment, metformin markedly decreased these weights. However, pioglitazone treatment (0.01, 0.02% in the HFD) did not have obvious effects on these parameters. Oral glucose tolerance test was carried out after 20-h fasting at 4 weeks post-treatment. Whereas metformin treatment (0.25, 0.5%) markedly improved glucose intolerance, pioglitazone treatment (0.02%) slightly improved this parameter. At 9 weeks, both metformin and pioglitazone markedly improved hyperglycemia and hyperinsulinemia. Metformin treatment also improved hyperleptinemia, whereas pioglitazone was ineffective. These results indicate that metformin reduces body weight gain and improves glucose intolerance in HFD-induced obese diabetic B6 mice.

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Keratinocyte growth factor-2 (KGF-2), also called fibroblast growth factor-10 (FGF-10), is a member of the fibroblast growth factor family. It plays a critical role in epithelial development and exerts its biological activities in a paracrine manner on the receptor FGFR2-IIIb. This study examined the function of topically applied KGF-2 in vivo on wound healing using a CO₂ laser, corneal epithelial wounded, rabbit model. Topically applied 25 μg/ml KGF-2 accelerated corneal epithelial wound healing, in contrast to the control, and reduced inflammation, stromal edema, and fibrosis. In addition, this factor also exhibited significant inhibition of corneal neovascularization. KGF-2 appears to be another important growth factor in the regulation of corneal epithelial wound healing.

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The voltage-gated K⁺ channel Kv1.5 was expressed in Chinese hamster ovary cells, and its interaction with fluvoxamine was studied using a whole-cell patch-clamp technique. Fluvoxamine reduced Kv1.5 whole-cell currents in a reversible, concentration-dependent manner, with an IC₅₀ value of 2.0 μM, and a Hill coefficient of 0.7. Fluvoxamine-induced inhibition of Kv1.5 caused a time-dependent blockade without modifying the kinetics of current activation. Fluvoxamine additionally inhibited Kv1.5 in a closed/resting or nonconducting state after deactivation. Inhibition increased steeply between −30 and 0 mV, corresponding to the voltage range for channel opening. Inhibition displayed an additional voltage dependence at voltages greater than 0 mV, consistent with an electrical distance of 0.16. Fluvoxamine slowed the deactivation time course and a tail crossover phenomenon was observed when the tail currents, recorded in the presence and absence of fluvoxamine, were superimposed. Inhibition of Kv1.5 by fluvoxamine was use-dependent. These results suggest that fluvoxamine strongly inhibits Kv1.5 currents and the inhibition of Kv1.5 by fluvoxamine is mixed channel-state dependent.

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The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl₄-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl₄-induced chronic liver injury in mice. The injection of CCl₄ into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl₄ treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

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Selective breeding was used to isolate hyperglycemic (ddY-H) or normoglycemic (ddY-L) mice that had been induced by fasting and refeeding. Serum glucose levels 12 h after 48 h-fasting were high in male ddY-H mice, but relatively low in male ddY-L mice compared with control ddY mice. Glucose tolerance was impaired in ddY-H mice maintained with standard chow pellets ad libitum at 12 weeks of age, and serum glucose and insulin levels were significantly increased after overnight fasting at 15 weeks of age. ddY-L mice indices did not differ from ddY mice indices, suggesting that insulin resistance is spontaneously induced in ddY-H mice. Increases in urinary excretion and urinary sugar accompanied by increased body mass were observed in all ddY-H mice, but not in ddY or ddY-L mice, at 27 weeks of age, indicating the induction of diabetic symptoms. Cross-mating between ddY-H and ddY-L mice was used to certify the genetic involvement in impaired glucose tolerance. This was not induced in mice born from male ddY-H and female ddY-L mice, or from female ddY-H and male ddY-L mice. In conclusion, ddY-H mice are a useful diabetic mouse model that show spontaneously induced insulin resistance followed by diabetic symptoms that are maintained by standard chow pellets. Their characteristics are recessively inherited, and ddY-L mice are an appropriate choice as control mice.

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The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract. The possibility of whether imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, modulates pacemaker activities in the ICC was examined using the whole cell patch clamp technique. Imatinib decreased the amplitude of pacemaker potentials in a dose-dependent manner in current-clamp mode. Because the effects of imatinib on pacemaker potentials were the same as those of pinacidil, we examined the effect of glibenclamide on ICC exposed to imatinib. The effects of imatinib on pacemaker potentials were blocked by glibenclamide. To see whether the production of prostaglandins (PGs) is involved in the inhibitory effect of imatinib on pacemaker potentials, we tested the effects of naproxen (a non-selective cyclooxygenase inhibitor) and AH6809 (a prostaglandin EP1 and EP2 receptor antagonist). Naproxen and AH6809 blocked the inhibitory effects of imatinib on ICC. Butaprost (an EP2 receptor agonist) showed the actions on pacemaker potentials in the same manner as imatinib. However, SC 19220 (an EP1 receptor antagonist) has no effects. To investigate the involvement of cAMP and protein kinase A (PKA) in the effects of imatinib on ICC, SQ 22536 (an inhibitor of adenylate cyclase) and mPKAI (an inhibitor of myristoylated PKA) were used. Both SQ-22536 and mPKAI blocked the imatinib-mediated inhibition of pacemaker potentials. However, the protein kinase C (PKC) inhibitors did not block the imatinib-mediated inhibition of pacemaker potentials. These results indicate that imatinib inhibits the pacemaker potentials of ICC by activating ATP-sensitive K⁺ channels and PKA-dependent, PKC-independent manner.

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Diabetes mellitus is a well known and important risk factor for cardiovascular diseases, including heart failure. A new model of Type 2 diabetes, Tsumura Suzuki Obese Diabetes (TSOD) mice, was introduced recently into the research field of diabetes. The cardiac functions of TSOD mice were studied in comparison with Tsumura Suzuki Non Obesity (TSNO, non-diabetic control) mice, for the first time. In vivo cardiovascular functions were measured by echocardiography and cardiac catheterization at 7, 12 and 18 months old. TSOD mice had no deterioration of cardiac function despite the long-term persistence of severe obesity, hyperglycemia, hyperinsulinemia and hyperlipidemia, including high density lipoprotein (HDL)-cholesterol. No histopathological abnormalities were observed in the heart of TSOD mice, while several histological abnormalities were observed in the pancreas and kidney of TSOD mice. To investigate vascular endothelium function at 7 months old, intravenous injection of acetylcholine (ACh; 1, 3, 10 μg/kg)- and N^G-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg)-induced mean blood pressure (BP) changes were used. ACh decreased whereas L-NAME increased BP, and no significant differences in BP changes were observed between TSOD and TSNO mice. Moreover, ACh-induced relaxation of the thoracic aortae isolated from TSOD and TSNO mice with intact endothelium were not significantly different. These findings suggest that vascular endothelial cells in TSOD mice are not impaired. It was clearly demonstrated that despite obvious diabetes, cardiac functions of TSOD mice were not impaired even at 18 months old.

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This study investigated the effect of human placenta extract (HPE) on cartilage degradation in vitro MG-63 cells, articular cartilage explants, and in vivo monoiodoacetate (MIA)-induced osteoarthritis (OA). Matrix metalloproteinase (MMP)-2 activity was measured in HPE-treated osteoblastic MG-63 cells. Articular cartilage explants in rabbit were cultured, and the degree of proteoglycan (PG) degradation was assessed by measuring the amount of glycosaminoglycan (GAG) released into the culture medium. Experimental osteoarthritis was induced by intra-articular injection of 3 mg MIA in rats. Beginning 14 d post-MIA injection, HPE was administered intra-articularly once a day for 14 d. The knee joints were assessed by roentgenography, histology, and gelatinase activity. HPE inhibited PG degradation in articular cartilage explants. HPE significantly reduced deformity of knee joints and suppressed the histological change in MIA-induced OA. HPE inhibited MMP-2 activity in MG-63 cells. MMP-2 and -9 activities were also reduced in the cartilages of HPE-treated knee joints. Our results indicate that HPE has therapeutic effects on OA by protecting cartilage.

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We have examined the involvement of serine/threonine protein phosphatase (PP) sensitive to okadaic acid (OA) in the antinociceptive effect of morphine in mice. The present study was performed to elucidate subcellular distribution and activity of OA-sensitive PPs in the brain when mice exposed to morphine. Subcutaneous administration of morphine (5 mg/kg) produced the antinociceptive effect with the maximum 30 min after its administration, evaluated by tail-pinch test. The antinociception was accompanied by an increment of activity in OA-sensitive PPs in the membrane fraction prepared from the whole brain of mice treated with morphine: The temporal profile of the morphine-induced increment of OA-sensitive PP activity was consistent with that of antinociceptive effects of morphine. The morphine-induced increase in OA-sensitive PP activity was dependent on the dose and attenuated by the concurrent administration of naloxone (1 mg/kg). To identify the subtype of OA-sensitive PPs in morphine-enhanced activity, we examined the level of PP2A and PP5, OA-sensitive PPs, in the subcellular fraction prepared from the whole brain of mice receiving morphine. Western blot revealed that morphine elicited the significant increase in the level of PP5, but not PP2A, in the membrane fraction, with the same peak time for the increment of PP5 as the antinociception. No significant change was observed in the level of OA-sensitive PPs in the cytosolic fraction at any examined time after morphine. These results suggest that the translocation of PP5 to the membrane fraction is, at least in part, involved in the antinociceptive effect of morphine in mice.

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We investigated the role of leukotriene (LT) B₄ in 5-lipoxygenase metabolite- and allergy-induced itch-associated responses using SA6541, an LTA₄ hydrolase inhibitor. Itch-associated responses were induced by intradermal injection of 5-hydroperoxyeicosatetraenoic acid (HPETE), a precursor of 5-lipoxygenase metabolites, and passive cutaneous anaphylaxis in ICR mice. By screening molecules related to arachidonic acid metabolism or pruritus, SA6541 was found to be a specific inhibiter of LTA₄ hydrolase. Pharmacokinetic studies confirmed the specificity of SA6541 at an oral dose of 100 mg/kg in mice. 5-HPETE induced scratching behavior, which was inhibited by SA6541 (100 mg/kg). However, SA6541 (100 mg/kg) hardly attenuated the 5-HPETE-induced increase in vascular permeability. Moreover, SA6541 (100 mg/kg) partially attenuated scratching behavior, but did not affect the increase in vascular permeability caused by passive cutaneous anaphylaxis. On the other hand, ketotifen fumarate, a histamine H1 antagonist, strongly inhibited the scratching behavior and the increase in vascular permeability caused by passive cutaneous anaphylaxis. These results suggest that LTB₄ is an endogenous itch mediator in the skin and is involved in the pruritus response in allergic reactions.

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Although donepezil, a potent acetylcholinesterase (AChE) inhibitor, has been used to treat Alzheimer's disease (AD) due to its neuroprotective effects, its mode of action to inhibit the growth of cancer cells is poorly understood. In the present study, we investigated the pro-apoptotic activities of donepezil in HL-60 human promyelocytic leukemia cells and the underlying molecular mechanism involved. It was found that donepezil induced the apoptosis of HL-60 and U937 cells in a dose- and time-dependent manner, as evidenced by the formation of DNA fragmentation and the accumulation of positive cells for Annexin V. In addition, the activations of caspase-8, -9, and -3 were significantly increased 36 h after donepezil treatment. Furthermore, the broad caspase inhibitor (z-VAD-fmk) blocked donepezil-induced apoptosis. In addition, donepezil was found to cause the loss of mitochondrial membrane potential (ΔΨ_m), to increase the release of cytochrome c to the cytosol, and to alter the expressions of Bcl-2 family proteins. Taken together, these results demonstrate for the first time that donepezil displayed an induction of apoptosis in HL-60 cells via a mitochondria-mediated caspase-dependent pathway.

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We investigated the effect of ammonium vanadate (vanadate) on ATP-induced increases in intracellular calcium ion level ([Ca²⁺]_i) of human umbilical vein endothelial cells (HUVEC) by fluorescence confocal microscopic imaging using the Ca²⁺-sensitive probe Calcium Green 1/AM. The ATP analogue 2-methylthio-ATP (2meS-ATP), at 10 μM, significantly increased the [Ca²⁺]_i of HUVEC, and this was abolished by 1 μM thapsigargin (a calcium pump inhibitor), whereas extracellular free calcium had no effect. Vanadate at 10 μM also significantly increased the [Ca²⁺]_i of HUVEC, which was abolished by 1 μM thapsigargin. However, vanadate at 1 μM did not exert such a significant effect. We thus examined the influence of ≤1 μM vanadate for 24 h on 2meS-ATP-induced increase in [Ca²⁺]_i. Vanadate significantly reduced the action of 2meS-ATP at 1 μM but not at 0.1 μM. Endogenously released ATP is known to induce various actions on endothelial cells. The present results suggest that vanadate exerts a regulatory influence on the function of vascular endothelial cells.

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Muscle disorders, such as muscular dystrophy, are associated with an increase in oxidative stress. Proposed treatments for muscular dystrophy, some in clinical trials, include gene therapy and muscle cell transplantation. In this study, we investigated the effects of idesolide, isolated from the fruits of Idesia polycarpa, on changes that occur in muscle disuse atrophy. We noted protective effects on oxidative stress response and HSP70 regulation. Pre-treatment with idesolide for 24 h maintained cell viability and decreased apoptosis in H₂O₂-treated C₂C₁₂ muscle cells. The idesolide pretreatment also increased intracellular HSP70 protein. Our results suggest that idesolide inhibits cell death through induction of HSP70 in C₂C₁₂ muscle cells. This work is the first to report that idesolide can regulate the decrease in HSP70 that occurs during skeletal muscle atrophy.

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CC chemokine receptor 4 (CCR4) is generally recognized as a preferential marker for T helper 2 cells, and we have previously reported morpholine-derivative CCR4 antagonists, RS-1154 and RS-1269. Here, we investigate the pharmacological profiles of a novel pyrimidine-derivative CCR4 antagonist, 2-{4-[2-(diethylamino)ethoxy]phenyl}-N-(2,4-difluorobenzyl)-5-fluoropyrimidin-4-amine (RS-1748), which showed potency to inhibit the bindings of [¹²⁵I]CCL17 and [³⁵S]GTPγS to human CCR4-expressing Chinese hamster ovary (CHO) cells with IC₅₀ values of 59.9 nM and 18.4 nM, respectively. Furthermore, RS-1748 inhibited ovalbumin-induced airway inflammation in guinea pigs at a dose of 10 mg/kg. These results indicate that RS-1748 would be a promising lead compound for developing a therapeutic agent against asthma.

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Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (n-3 PUFAs), is an essential polyunsaturated fatty acid in the central nervous system, and possesses many physiological functions in neurodegenerative diseases. Previously, there are some reports that n-3 PUFAs contribute to pain relief. As the antinociceptive effect of DHA alone has not been reported, this study examined the antinociceptive effect of DHA on various pain stimuli. To evaluate the antinociceptive effect of DHA on thermal and chemical nociception, we employed the tail flick test, acetic acid writhing test and formalin test in mice. DHA was orally administrated at 5, 15 and 25 mmol/kg at 30 min before measurement. DHA administration dose-dependently exerted an antinociceptive effect against thermal and chemical stimulation in comparison to the control olive oil administration. These effects of DHA were abolished when mice were pretreated with naloxone, an opioid receptor antagonist. These findings suggest that DHA has opiod receptor-mediated pain control activities, and may provide valuable information towards an advanced therapeutic approach for pain control.

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Cancer gene therapy with adenovirus vectors (Adv) is limited to local administration because systemic administration of Adv produces a weak therapeutic effect and severe side effects. Previously, we generated a dual cancer-specific Adv system by using Adv covalently conjugated to polyethylene glycol (PEG) for transductional targeting and the telomere reverse transcriptase (TERT) promoter as a cancer-specific promoter for transcriptional targeting (PEG-Ad-TERT). We demonstrated that systemic administration of PEG-Ad-TERT showed superior antitumor effects against lung metastatic cancer with negligible side effects. Here, we investigated the therapeutic efficacy of systemic administration of PEG-Ad-TERT for the treatment of primary tumors. We first evaluated the transgene expression of PEG-Ad-TERT containing the luciferase gene (PEG-Ad-TERT/Luc) in primary tumors. Systemic administration of PEG-Ad-TERT/Luc resulted high transgene expression, similar to that observed in tumors for the conventional cytomegalovirus (CMV) promoter-driven Adv containing the luciferase gene (Ad-CMV/Luc). By comparison, transgene expression was 2500-fold lower than that of Ad-CMV/Luc in liver. We then examined the therapeutic effect of systemic administration of PEG-Ad-TERT containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk) for the treatment of primary tumors. We showed that PEG-Ad-TERT/HSVtk produced a notable antitumor effect against primary tumors with negligible side effects. These results demonstrated that PEG-Ad-TERT can be regarded as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both metastatic and primary tumors.

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Passionflower (Passiflora incarnata L.) is used in phytotherapy as a mild sedative and anxiolytic agent. In the literature it is clear this plant shows considerable qualitative and quantitative variability with respect to its content of C-glycosyl flavones, some of which are used as marker compounds for extracts. Analysis of plant material cultivated in Australia revealed two chemically distinct groups; hence an investigation was carried out to determine whether distinct intraspecific chemotypes exist in this species. Eleven P. incarnata samples were analysed by HPLC, LC-MS and two different TLC methods. The samples fell into two distinct groups with respect to their C-glycosyl flavone profile, with little within-group variation. One chemotype was dominated by isovitexin and schaftoside/isoschaftoside, as is most widely reported in the literature for this species. The other chemotype was characterized by a high level of swertisin, with low levels of schaftoside/isoschaftoside. The two chemotypes are readily identified by both HPLC and TLC. Although the compounds responsible for the therapeutic activity of P. incarnata are yet to be identified, phytomedicines should be made with the accepted isovitexin chemotype until the pharmacological implications of chemotypical differences are understood.

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Oxidized low-density lipoprotein (oxLDL) and reactive oxygen species (ROS) play key roles in the early stage of atherosclerosis. Nitric oxide (NO) and ROS are responsible for regulation of the transcriptional pathways of nuclear Factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), key regulators of cellular inflammatory and immune responses. Previously, we examined LDL-antioxidant activities of the nine flavonoids isolated from Sophora flavescens. Among these, two lavandulyl flavonoids, kurarinone (1) and kuraridin (2) inhibited inducible nitric oxide synthase (iNOS)-dependent NO production and ROS generation, and suppressed remarkably the expression of inflammatory cytokines, CCL2, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and iNOS in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Moreover, compounds 1 and 2 attenuated NF-κB activation by inhibition of IκBα proteolysis and p65 nuclear translocation, as well as phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases.

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The effects of heat-processed ginseng (HPG) and ginsenoside 20(S)-Rg₃ on the progression of renal damage in type 2 diabetic rats were investigated. Twenty-two-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were divided into 4 orally administered groups: vehicle (diabetic control), HPG water extract (100 mg/kg) and 20(S)-Rg₃ (5, 10 mg/kg). Non-diabetic Long-Evans Tokushima Otsuka (LETO) rats were used as a normal group. OLETF rats showed markedly higher blood glucose, triglyceride, and total cholesterol levels than those of LETO rats. The elevated blood glucose level of OLETF rats was significantly lowered by 20(S)-Rg₃ administration. The elevated serum triglyceride and total cholesterol levels were significantly reduced by the administrations of HPG and 20(S)-Rg₃. The serum levels of thiobarbituric acid-reactive substance, an index of lipid peroxidation, were markedly increased in OLETF compared to LETO rats, but it was significantly reduced by HPG and 20(S)-Rg₃ administrations. The urinary protein level, an indicator of advanced diabetic nephropathy, of OLETF rats was 4.4 times higher than in LETO rats, but it was reduced significantly by the administrations of HPG and 20(S)-Rg₃. Creatinine clearance of OLETF rats was significantly increased after HPG and 20(S)-Rg₃ administrations. The elevation of inducible nitric oxide synthase and N^ε-(carboxymethyl)lysine protein expressions in renal tissues of OLETF rats was prevented by 20(S)-Rg₃ administration. This study provides scientific evidence that 20(S)-Rg₃ prevents the progression of renal damage and dysfunction in type 2 diabetic rats via inhibiting oxidative stress and advanced glycation endproduct formation.

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2′-Deoxycytidine (dCyd), a pyrimidine nucleoside found at high concentrations in the plasma of cancer patients with a poor prognosis after chemotherapy, is considered to be a biomarker for breast cancer. 5-Fluorouracil (5FU) is a nucleoside analog and is used as an anti-tumor agent in patients whose plasma dCyd concentrations are increased. Because both dCyd and 5FU are pyrimidine analogues, it is possible that they have pharmacokinetic/ pharmacodynamic interaction, by which the anti-cancer efficacy of 5FU would be reduced. Here, we examined the effects of dCyd on the cytotoxicity of 5FU on mouse myeloma SP2/0-Ag14 (SP2/0) cells lacking hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) and RH4 hybridomas with HGPRT under asynchronized conditions. The reduced cell viability by 5FU was restored by co-, but not pre-, treatment of dCyd in both SP2/0 and RH4 cells, but this effect in the former tended to be greater than that in the latter, suggesting a possible involvement of HGPRT in the interaction, although this might not be a major mechanism. Moreover, dCyd administration to SP2/0 myeloma-bearing mice tended to shorten their 5FU-induced prolonged survival in vivo. Collectively, these results indicate that dCyd decreases the anti-tumor efficacy of 5FU and that a metabolic pathway via HGPRT is involved partially in this interaction. The evaluation of dCyd as a biomarker is believed to provide valuable information for effective and safe chemotherapy with 5FU.

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Asthma is characterized by chronic eosinophilic inflammation and hyperresponsiveness of the airways. We hypothesized that thalidomide, which has numerous immunomodulatory properties, may have anti-inflammatory effects in allergic asthma. BALB/c mice sensitized and challenged with ovalbumin (OVA) were treated orally with thalidomide (30, 100, or 300 mg/kg) or a vehicle. When thalidomide was administered to OVA-challenged mice, the number of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly decreased. The numbers of inflammatory cells other than eosinophils were not reduced by thalidomide. Thalidomide inhibited the elevated levels of interleukin-5 (IL-5) and tumor necrosis factor-α (TNF-α) in BALF by OVA challenges. Histological analysis of the lung revealed that both the infiltration of inflammatory cells and the hyperplasia of goblet cells were significantly suppressed by thalidomide treatment. Furthermore, thalidomide significantly inhibited the response to methacholine induced by OVA challenges. Taken together, thalidomide treatment decreased airway inflammation and hyperresponsiveness in a murine model of allergic asthma. These results might provide an opportunity for the development of novel therapeutics to treat severe asthma.

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To determine if gene expression profiling of whole blood cells is a useful source of markers for the early diagnosis of the onset of type 2 diabetes, we examined gene expression profiling of whole blood cells and type 2 diabetes-related organs, such as liver, adipose tissue, and skeletal muscle, of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. At the age of 6 weeks, RNA was isolated from tissues of fasted OLETF and control Long-Evans Tokushima Otsuka (LETO) rats. Gene expression was analyzed using the Agilent rat oligo microarray. Gene ontology analysis showed that gene expression of biologically meaningful groups of genes in liver, adipose tissue, and skeletal muscle, which are involved in the pathogenesis of type 2 diabetes, differed between OLETF and LETO rats. Three hundred genes of whole blood cells were differentially expressed. Four out of these 300 genes were related to the insulin-signaling pathway and 57 out of 300 genes were up- or down-regulated in at least one tissues in OLETF rats. These results support our hypothesis that gene expression profiling of whole blood cells might be a useful source of markers to predict the onset of type 2 diabetes.

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Pharmacokinetic/pharmacodynamic (PK/PD) modeling and simulation enable the prediction of the effect of a medication in various situations in clinical practice. The aims of this study were to predict the relationships between the effect of rosuvastatin and various factors such as poor compliance, and morning and evening dosages, as well as the change in the pharmacokinetics of rosuvastatin. We characterized the PK/PD model of plasma mevalonic acid (MVA) profiles after rosuvastatin administration and simulated the plasma MVA concentration in various dosage regimens. The plasma rosuvastatin and MVA concentrations reported by Martin et al. were used as the source of PK/PD modeling data. For each simulation, a summary parameter, the area under the plasma MVA concentration–time curves for 24 h in the steady state (AUEC₂₄), was used to characterize the time course of each endpoint. To estimate the influence of PK parameters on rosuvastatin effects, the AUEC₂₄ reduction ratio of baseline levels was simulated from the 0.33—3.0-fold value of each PK parameter estimate. The AUEC₂₄ reduction ratio was 7.7% lower after morning administration than after evening administration. The changes in the PK parameters more prominently affected the AUEC₂₄ reduction ratio after morning administration than after evening administration. The simulated plasma MVA concentrations almost reached their baseline levels in the case of patients who forgot to take rosuvastatin. These results suggest that the parameters can be used to determine the effective rosuvastatin dosage regimen.

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Monoclonal antibodies are being widely used for the treatment of various diseases. Microparticle formation in high-concentration protein solutions is a major problem during the manufacture of therapeutic monoclonal antibodies, because aggregation leads to fouling of aseptic filters and may lead to an immunogenic reaction in patients. We found that stirring using a traditional bottom-magnetic type stirrer results in extensive and sustained formation of 500-nm diameter protein microparticles arising from shear stress on protein molecules. The antibody solution stirred for only 5 min using this type of stirrer exhibited significant fouling of aseptic filter membranes. In contrast, a top-entering type stirrer did not lead to the formation of microparticles, and the solution did not exhibit membrane fouling even after 30 min of stirring. We conclude that a top-entering type stirrer is more suited for the manufacture of concentrated therapeutic monoclonal antibody solutions.

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