Interleukin (IL)-6 cDNA was originally cloned as a terminal B cell differentiation factor into antibody-producing plasma cells. This revealed that it is a multifunctional cytokine that acts on a variety of cells. From the clinical viewpoint, it is especially important that IL-6 acts on hepatocytes to induce acute-phase reactants, including C-reactive protein, serum amyloid A protein, and fibrinogen, and to decrease serum albumin levels. Very recently, this cytokine has been found to enhance the synthesis of a peptide called hepcidin in the liver which regulates iron recycling, resulting in anemia due to hypofferemia. It has also been shown that IL-6 is responsible for various clinical symptoms, including the appearance of autoantibodies, fatigue, anemia, anorexia, fever, and increases in the erythrocyte sedimentation rate, all of which develop in patients with various chronic autoimmune inflammatory diseases. In practice, blocking the IL-6 signaling pathway with a recombinant humanized anti-IL-6 receptor antibody, tocilizumab (TCZ), has dramatically improved all the signs and symptoms of these patients. A study in mice demonstrated that IL-6 promotes the development of a new type of T-helper cells called Th17 cells that impact the pathogenesis of autoimmune diseases. This suggests that TCZ is not only an antiinflammatory agent but also might affect basic autoimmunity. In this review, recent advances in the immunobiology of interleukin-6 related to immune-mediated diseases are discussed.
Metabolism by catechol-O-methyltransferase (COMT) is one of the inactivation pathways of catecholamines (CAs), which are important hormones in regulating blood pressure both in central and in peripheral sympathetic nerve endings. We have reported the rapid determination method of COMT activity using high-performance liquid chromatography (HPLC)-fluorescence detection. In the present study, we applied the method to brain tissues, cerebral cortex, cerebellum, hypophysis and hypothalamus. COMT activities were assessed by measuring normetanephrine with the use of norepinephrine as an endogenous substrate. We examined the COMT activities in the brains of Dahl salt-sensitive (DS) rats given normal-salt or high-salt diet for 13 weeks, and found that membrane-bound COMT activities in the cerebral cortex were significantly reduced in high-salt loaded DS rats compared with normal-salt loaded DS rats.
In our efforts to find new skin lightening agents, piceatannol (PICE) was investigated for its antioxidative property and ability to inhibit melanogenesis. In this study, PICE's effect on inhibition of mushroom tyrosinase, and tyrosinase inhibiting activity and melanin content were assessed utilizing the B16F10 melanoma cell (B16 cell) culture system. Results indicated that PICE has a strong antityrosinase activity (IC50=1.53 μM). To evaluate the relative efficacy of PICE compared to other tyrosinase inhibitors, its inhibitory effect was compared and showed that PICE was significantly stronger than kojic acid (IC50=50.1 μM) and resveratrol (IC50=63.2 μM). Furthermore, PICE was shown to down-regulate melanin content. To document PICE's antioxidative property, which is known to influence melanogenic activity, we assessed reactive species (RS) generation, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in these B16 cells. The results showed that PICE suppressed RS generation and enhanced the GSH/GSSG ratio. In conclusion, our results indicated that the antimelanogenic action of PICE is likely exhibited by the combined effect of PICE's antioxidative property and its ability to suppress RS generation while increasing the GSH/GSSG ratio.
The basic disaccharide structure recognized by galectin family members is the lactosamine-like structure Galβ1-4(3)Glc(NAc). In galectins, eight highly conserved amino acid residues participate in the recognition of this basic structure. Each galectin seems to mediate diverse biological functions due to recognition of different modifications of the basic disaccharide Galβ1-4(3)Glc(NAc), but there is very little information about which amino acid residue in galectin is responsible for recognizing these modifications. The 32-kDa galectin LEC-1 of the nematode Caenorhabditis elegans is composed of two domains, each of which is homologous to vertebrate 14-kDa-type galectins. Although both lectin domains have an affinity for N-acetyllactosamine (Galβ1-4GlcNAc)-containing, N-linked, complex-type sugar chains, the N-terminal lectin domain of LEC-1 recognizes blood group A saccharide (GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAc), whereas this saccharide is only poorly recognized by the C-terminal domain. Here, we used a combination of site-directed mutagenesis of the N-terminal lectin domain of galectin LEC-1 and an analysis of the sugar-binding profile by frontal affinity chromatography to identify the amino acid residues important for this recognition. Our results indicate that Thr41 in the N-terminal lectin domain of LEC-1 is important for its affinity for A-hexasaccharide.
Hypoxia-inducible factor-1 (HIF-1) is a main regulator of metabolic adaptation to hypoxia. HIF-1α is induced by hypoxia, or by hypoxia-mimicking reagents, such as desferrioxamine (DFX), under a normoxic condition. A xanthine oxidase inhibitor, 6-formylpterin (6FP), is reported to exert its functions on reactive oxygen species (ROS) modulation. In this study, we investigated the effect of 6FP on HIF-1α expression under a DFX-treated or hypoxic condition. 6FP decreased HIF-1α expression at the protein level, but not at the mRNA level, in a dose-dependent manner, and this suppressive effect was reversed by the antioxidant, N-acetyl-L-cysteine (NAC). Furthermore, the ROS generated by 6FP was reversed with NAC coincubation. These findings suggest that intracellular ROS generated by 6FP decreased the HIF-1α protein accumulation under a DFX-treated or hypoxic condition.
Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has reproductive and developmental toxicity as an estrogen antagonist, we investigated the effects of TCDD on osteogenesis in rat skeleton and the human female-responsive osteoblastic osteosarcoma cell line SaOS-2. Rat fetuses were exposed to 5, 10, or 15 μg/kg TCDD on gestation day (GD) 10. TCDD dose-dependently induced single or multiple rat fetal skeletal development malformations in vivo. In vitro, 10 nM TCDD significantly inhibited cell proliferation in the presence of 1 μM 17-β-estradiol (E2) in SaOS-2 cells. Insulin-like growth factor binding protein 6 (IGFBP-6), as a crucial regulator in IGF system, plays an important role in osteogenesis and bone function. TCDD (15 μg/kg) induced a dramatic 3-fold increase in IGFBP-6 mRNA expression in rat fetal calvaria on GD 21. On the other hand, the concurrent treatment of 10 nM TCDD and 1 μM E2 resulted in a significant increase in IGFBP-6 mRNA and protein after 24 h in SaOS-2 cells, but TCDD and (or) E2 had no effect on the mRNA level of cytosolic aromatic hydrocarbon receptor. The functional estrogen-responsive element (ERE) [5′-CCT TCA CCT G-3′] (−9 to +1) in the IGFBP-6 promoter region was identified in this study for the first time as the ER genomic binding site. Collectively, these results suggest that TCDD can alter the expression of IGFBP-6 gene and exerts growth-inhibitory effects on osteogenesis. In addition, TCDD exhibits an anti-estrogenic effect through its interference with the binding of activated estrogen-liganded ER to the functional ERE in IGFBP-6 gene promoter.
A systematic analysis to examine the effects of peroxisome proliferator-activated receptor (PPAR)α agonists on the expression levels of all the nutrient/drug plasma-membrane transporters in the mouse small intestine was performed. Transporter mRNAs that were induced or repressed by two independent PPARα-specific agonists were identified by a genome-wide microarray method, and the changes were confirmed by real-time PCR using RNA isolated from the intestines and livers of wild-type and PPARα-null mice. Expression levels of seven nutrient/drug transporters (Abcd3, Octn2/Slc22a5, FATP2/Slc27a2, Slc22a21, Mct13/Slc16a13, Slc23a1 and Bcrp/Abcg2) in the intestine were up-regulated and the expression level of one (Mrp1/Abcc1) was down-regulated by PPARα; although the previously report that the H+/peptide co-transporter 1 (Pept1) is up-regulated by PPARα was not replicated in our study. We propose that the transport processes can be coordinately regulated with intracellular metabolism by nutrient nuclear receptors.
Human parvovirus B19 is a clinically important pathogen in both children and adults. In adults, it frequently causes acute and chronic arthritis, which may be related to persistent infection. The effect of the capsid of human parvovirus B19 on monocytes, which are thought to be responsible for the first line of defense against parvoviral infection, is not well understood. In this study, we investigated changes in mRNA expression levels of several immunoregulatory cytokines in monocytic cells after treatment with the B19 capsid. When human monocytic cell line THP-1 cells were treated with the B19 capsid, the expression of tumor necrosis factor alpha (TNF-α) mRNA was suppressed independently of transforming growth factor beta (TGF-β) mRNA. In contrast, the level of mRNA for interleukin-1 alpha (IL-1α) remained unchanged, and that for interleukin-1 beta (IL-1β) was slightly increased after the capsid treatment. Flow cytometry demonstrated that THP-1 cells treated with B19 capsid showed no differences in surface expression of CD11a, CD16 and CD33, as compared with control cells. These findings that B19 capsid antigen did not promote positive responses for production of TNF-α and IL-1α may provide insight into the mechanisms of persistent infection of human parvovirus B19 and the systemic viral spread via bloodstream.
The simplest amino acid, glycine, is important in protein composition and plays a significant role in numerous physiological events in mammals. Despite the inhibitory effect of glycine on spontaneous melanogenesis in B16F0 melanoma cells, the details of the underlying mechanisms remain unknown. The present study was conducted to investigate the further effects and the mechanisms of inhibitory effect of glycine on melanogenesis using B16F0 melanoma cells and hair follicle melanogenesis in C57BL/6J mice. Treatment with glycine (1—16 mM) for 72 h inhibited α-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in a concentration-dependent manner without any effects on cell proliferation in B16F0 melanoma cells. Treatment with kojic acid (2.5 mM) for 72 h also inhibited α-MSH-induced melanogenesis in B16F0 melanoma cells. The highest dose of glycine inhibited the α-MSH-induced increment of tyrosinase protein levels in B16F0 melanoma cells. In hair follicle melanogenesis in C57BL/6J mice, treatment with glycine (1250 or 2500 mg/kg, i.p.) for 5 d prevented the decrement of L* and C* values and inhibited the increment of tyrosinase protein levels and melanin content within the skin. Treatment with hydroquinone (100 mg/kg, i.p.) for 5 d had a similar hypopigmenting effect to that of high dose glycine. These results suggest that glycine has an inhibitory effect on melanogenesis that is mediated by down-regulation of tyrosinase protein levels, leading to a hypopigmenting effect in C57BL/6J mice.
Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker activities by ginseng total saponins in the interstitial cells of Cajal (ICC) using the whole cell patch-clamp technique. Externally applied ginseng total saponins (GTS) produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker currents, but only treatment with flufenamic acid inhibited the GTS-induced tonic inward currents. The tonic inward currents induced by GTS were not inhibited by the intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca2+-free solution, with U-73122, an active phospholipase C inhibitor, and with thapsigargin, a Ca2′-ATPase inhibitor of the endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the GTS-induced action. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block the GTS-induced effects on the pacemaker currents. These results suggest that ginsenosides modulate the pacemaker activities of the ICC, and the ICC can be targets for ginsenosides, and their interaction can affect intestinal motility.
Korean mistletoe lectin (KML) is one of the major active components in Viscum album var. (coloratum), displaying various biological effects such as anti-tumor and anti-metastatic activities. Even though it has been shown to boost host immune defense mechanisms, the immunomodulatory effects of KML on specific immune responses mediated by macrophages have not been fully elucidated. Therefore, in this study, we aimed to demonstrate KML's regulatory roles on macrophage-mediated immune responses. KML clearly blocked lipopolysaccharide (LPS)-induced events [expression of interleukin (IL)-10, nitric oxide (NO) production and phagocytic uptake], and suppressed the normal expression levels of IL-10 (at 2 ng/ml) and tumor necrosis factor (TNF)-α (at 10 ng/ml). In contrast, (1) the expression of cytokine (TNF-α) and (2) the generation of reactive oxygen species (ROS) induced by LPS were significantly up-regulated with KML co-treatment. In addition, KML itself increased the mRNA levels of IL-3 and IL-23; phagocytic uptake; the surface levels of co-stimulatory molecules (CD80 and CD86), pattern recognition receptors (PRRs) [such as dectin-1 and toll like receptor (TLR)-2] and adhesion molecules [β1-integrins (CD29) and CD43]; and CD29-mediated cell adhesion events. Finally, according to co-treatment of D-galactose with KML under LPS-induced NO production conditions, KML inhibition seems to be mediated by binding to proteins with D-galactose. Therefore, these data suggest that KML may participate in regulating various macrophage-mediated innate and adaptive responses via binding to surface protein with D-galactose and that some of these may deserve in KML's therapeutic activities such as anti-tumor and anti-microbial effects.
The effects of esculetin (6,7-dihydroxycoumarin) and its 6-glycoside, esculin, on 8-oxo-2′-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, 1,2-dimethylhydrazine (DMH), were examined in the colons of male Fischer 344 rats. Animals were given water containing esculetin or esculin for 7 d before subcutaneous injection of DMH (20 mg/kg body wt), killed 24 h after DMH treatment, and the levels of thiobarbituric acid reactive substances (TBARS) and 8-oxodG in the colons were determined. Both esculetin and esculin suppressed significantly the DMH-induced increases in 8-oxodG and TBARS in rat colon mucosa. We further investigated the modifying effect of esculin intake on the development of DMH-induced colonic aberrant crypt foci (ACF). Animals were given DMH once a week for 4 weeks to induce ACF. They then received water containing esculin ad libitum for 5 weeks (initiation phase) or 11 weeks after DMH treatment (post-initiation phase). Animals in the positive control group received tap water throughout the experiment. At the end of the experiment (16 weeks), the ingestion of esculin during the initiation phase significantly reduced the incidence of gross tumors, the number of ACF per rat and the mean number of AC per focus, while the esculin treatment during the post-initiation phase significantly decreased only the number of ACF per rat. These results suggest that esculin intake has an inhibitory effect on DMH-induced oxidative DNA damage and carcinogenesis in rat colons.
It has been reported that emodin is able to promote gastrointestinal motility and stimulate large intestinal water secretion; however, the mechanism is still not clear. The aim of the present study is to examine the effects of emodin on the rat colonic transepithelial ion transport and the underlying mechanism. The study was carried out by means of the short circuit current (ISC) recording. Basolateral application of emodin induced a concentration-dependent ISC increase, and the EC50 was 76.0 μmol/l. Pretreatment with epithelial Na+ channel blocker, amiloride (10 μmol/l), did not affect the ISC responses elicited by emodin, but removal of extracellular Cl− or apical pretreatment with Cl− channel blocker, glibenclamide (1 mmol/l) inhibited emodin-elicited ISC responses by 76.3% and 83.8% respectively. Inhibiting basolateral Na+–K+–2Cl− cotransporter (NKCC) with bumetanide (100 μmol/l) decreased emodin-induced ISC from 118.1±6.7 μA/cm2 to 16.7±2.0 μA/cm2, which was reduced by 85.9%. Basolateral pretreatment with neuronal Na+ channel blocker tetrodotoxin (TTX) (1 μmol/l) did not affect emodin-induced ISC increase, but pretreatment with indomethacin (10 μmol/l) alone or with both TTX and indomethacin significantly decreased emodin-induced ISC increase by 88.4 and 81.2%, respectively. The present study demonstrated that emodin was able to stimulate rat colonic epithelial Cl− secretion, which was predominantly mediated by endogenous prostaglandin release.
Although the fruit of Nandina domestica THUNBERG (ND) has been used to treat respiratory disorders such as coughing and breathing difficulty in Japan for many years, very little is known about mechanisms underlying its action. In the present study, we investigated effects of the crude extract from ND (NDE) and one of its constituents, nantenine, on contractile responses in isolated guinea pig tracheal ring preparations. In normal experimental condition, guinea pig trachea remained tonically contracted during the resting state, and addition of NDE (1 mg/ml) caused a relaxation of tracheal smooth muscles, but had little effect on the responsiveness of trachea to acetylcholine. The basal, tonic contraction was abolished by the presence of atropine and indomethacin. In this condition, NDE at 0.1—1 mg/ml inhibited histamine-induced contraction in both competitive and non-competitive manners. NDE at 0.01—1 mg/ml inhibited serotonin-induced contraction in a competitive manner. Nantenine (2—20 μM) did not affect histamine-induced contraction, and slightly inhibited serotonin-induced contraction. These results suggest that NDE has inhibitory effects on tracheal smooth muscle contraction, and nantenine cannot account solely for this effect of NDE.
Cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite used to induce remission in acute leukemia, but cellular resistance to Ara-C reflects a poor prognosis in cancer chemotherapy. To further investigate the mechanisms of resistance to Ara-C, we have established Ara-C-resistant NALM-6 cells. The activation of nuclear factor κB (NF-κB) was accompanied by the acquisition of Ara-C resistance. Telomerase activity has also increased with the acquisition of Ara-C resistance. The expression of Bid, Bax, or p53 proteins have been shown to increase correlated with the acquisition of Ara-C resistance. In contrast to the increase in these proteins, Bcl-2, Bcl-x, and Bag-1 proteins remained unchanged with the acquisition of Ara-C resistance. Fas expression increased with the acquisition of Ara-C resistance in the late stage. The induction of apoptosis and reduction of cell viability by cytotoxic anti-Fas antibody was more susceptible in resistant cells than parental cells. In conclusion, this report has shown that resistance to Ara-C up-regulates the activation of NF-κB, telomerase activity and Fas expression.
In the present study, we have examined the hypoglycemic effect of maslinic acid (MA) in KK-Ay mice, an animal model of genetic type-2 diabetes. MA (10 mg/kg body wt) reduced the blood glucose levels in KK-Ay mice at 4 h after a single oral dose. KK-Ay mice receiving MA at daily dosages of 10 mg/kg and 30 mg/kg for 2 weeks showed a significant reduction in the blood glucose levels. Furthermore, the results also showed that MA might modulate glucose metabolism partially through reducing insulin resistance in KK-Ay mice. Taken together, MA may hold great promise as a natural therapeutic agent for treatment of type 2 diabetes.
The anti-hyperglycemic action of Stephania tetrandra Radix (Stephania) is potentiated by Astragalus membranaceus BUNGE Radix (Astragali) in streptozotocin (STZ)-diabetic ddY mice (Tsutsumi et al., Biol. Pharm. Bull., 26, 313 (2003)). Fangchinoline (0.3—3 mg/kg), a main constituent of Stephania, decreased the high level of blood glucose and increased the low level of blood insulin in STZ-diabetic mice. Here, we investigated the combined effects of fangchinoline with isoflavone or isoflavonoid components (formononetin, calycosin and ononin) of Astragali on the hyperglycemia and hypoinsulinemia of STZ-diabetic mice. Formononetin, calycosin and ononin (0.03—0.1 mg/kg) alone did not affect the blood glucose or blood insulin level of the diabetic mice. Formononetin and calycosin (0.03—0.1 mg/kg) potentiated the anti-hyperglycemic action of fangchinoline (0.3 mg/kg), but ononin did not. Formononetin (0.1 mg/kg) facilitated the fangchinoline-induced insulin release, and calycosin (0.1 mg/kg) also facilitated it, though without statistical significance. In conclusion, the combined effect of fangchinoline with formononetin and calycosin on hyperglycemia in the diabetic mice accounted well for the therapeutic effect of the combination of Stephania with Astragali in Boi-ogi-to. The anti-hyperglycemic action of formononetin appeared to be due to its potentiating action on insulin release. Our strategy for studying combinations of crude drugs and their components in Kampo medicine has uncovered new potentiating effects of formononetin and calycosin on the anti-hyperglycemic action of fangchinoline in STZ-diabetic mice.
Objective: To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with β-thalassemia major. Methods: YSSXG was given orally to 156 patients with β-thalassemia in GuangXi Autonomous Region (the high incidence area of β-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3—6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. Results: Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly meliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase γ/(β+γ) globin ratio, both γ-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG. Conclusion: YSSXG had obviously effects in treating β-thalassemia by unlocking γ-gene, increasing the γ-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of β-thalassemia with Traditional Chinese Medicine.
In the present study, we investigated the changes of sleep parameters in rats with chronic constriction injury (CCI) under aversive conditions. The electroencephalogram (EEG) in the frontal cortex of CCI rats and electromyogram (EMG) were measured over 6 h by placing rats on sandpaper as an aversive condition, to compare with rats placed on sawdust. Six days after CCI surgery, the rats exhibited significant mechanical allodynia, and also had neuropathic pain. When rats were placed on sawdust, no significant difference was observed between the CCI group and sham-operated control group in sleep latency, total waking time, total non-REM sleep time and total REM sleep time. On the other hand, when CCI rats were placed on sandpaper, a significant increase was observed in sleep latency and total waking time compared with the sham group; however, no significant difference was observed in the total non-REM sleep time and total REM sleep time between these two groups. These results indicate that an important factor of sleep disturbance in CCI rats is not only damage to the nerves but also being under aversive conditions. In addition, it was found that CCI rats placed on sandpaper as an aversive condition can serve as a new sleep disturbance model.
Human fetal liver (HFL) cell culture was initiated from a pool of six normal human liver tissues. The proliferation and viability of HFL cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay, and the cells increased by more than 100-fold by culture for 15 d. The levels of expression of albumin (ALB), hepatocyte nuclear factor 4α, hepatocyte growth factor, CYP3A4, CYP3A5, and CYP3A7 mRNAs in HFL cells increased with culture period, while that of α-fetoprotein (AFP) mRNA decreased gradually. In HepG2 cells, however, the expression levels of ALB and AFP mRNAs were not changed, and the levels of expression of CYP3A4, CYP3A5, and CYP3A7 mRNAs decreased gradually. The mRNA expression of major CYP isoforms including CYP3As, i.e., CYP1A2, CYP2A6, CYP2B6, CYP2C (2C9 and 2C19), CYP2D6, and CYP2E1, could be detected in HepG2 cells. With the exception of CYP1A2, all of the CYP mRNAs expressed in HepG2 cells were detected in HFL cells. In HFL cells, CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA expression was increased to a level 3-fold greater than control by DEX. On the other hand, CYP3A4, CYP3A5, and CYP3A7 mRNA expression levels in HepG2 cells were increased from 2- to 3-fold by treatment with DEX and RIF. Pregnane X receptor mRNA was expressed in HepG2 cells, but not HFL cells. These results indicate that the character of HFL cells with regard to CYP expression was different from that of HepG2 cells.
Sinoaortic denervated (SAD) rats present arterial pressure lability without sustained hypertension. We investigated the relation between sinoaortic denervation and the occurrence of oscillatory contractions in SAD rat aortas, as well as the effect of various K+ channel modulators on these oscillations. Aortas were removed and concentration–effect curves to phenylephrine (0.01 to 10 μM) were constructed in arteries from SAD and Sham-operated rats in order to verify the occurrence of oscillations. We also evaluated the effects of various K+ channel modulators on these oscillations. Only SAD rat aortas exhibited oscillatory contractions. Tetraethylammonium increased the frequency (28.5±3.5 to 41.5±4.5 counts/5 min) and amplitude (0.435±0.07 to 0.630±0.09 g) of the oscillations. Apamin and 4-aminopyridine did not alter the oscillations. Barium chloride converted the oscillatory contractions to a tonic contraction. Pinacidil rapidly blocked the oscillatory contractions and glibenclamide evoked reduction in amplitude from 0.410±0.07 to 0.180±0.06 g. Iberiotoxin increased the frequency of oscillatory contractions (from 28.0±3.5 to 51.5±7.5 counts/5 min) but decreased the amplitude (from 0.410±0.08 to 0.195±0.2 g). Our results demonstrate that SAD rat aortas exhibit oscillatory contractions and K+ channels, mainly KATP and BKCa, play a dominant role in these oscillations.
In this study, we examined the immunoregulatory roles of L-cycloserine (L-CS), a sphingolipid metabolism regulator with inhibitory activity of serine palmitoyltransferase (SPT), on immune responses mediated by monocytes/macrophages and T cells. Mitogenic responses of splenic lymphocytes induced by LPS, PHA, and Con A were very strongly suppressed by L-CS with IC50 values ranging from 0.5 to 1 μM. In contrast, this compound less strongly blocked IL-2-induced CD8+ CTLL-2 cell proliferation with an IC50 value of 540 μM. Interestingly, L-CS enhanced the number of IL-4-producing helper T cells, indicating the favored induction of Th2 condition. Although tumor necrosis factor (TNF)-α and nitric oxide (NO) production was not altered under 10% FCS condition, U937 cell–cell adhesion as well as the surface level of adhesion molecules (CD29 and CD98) were significantly suppressed by L-CS. In particular, reduced serum level (5%) under L-CS treatment strongly enhanced the production of TNF-α and the inhibitory potency of NO production and cell adhesion. Finally, sphingolipids (D-sphingosine and DL-dihydrosphingosine) did not remarkably abrogate L-CS-mediated T cell proliferation. Therefore our data suggest that de novo sphingolipid metabolism may represent an important aspect of immunomodulatory activities mediated by T cells and macrophages/monocytes, depending on serum level.
Cisplatin is one of the most effective chemotherapeutic agents and plays a major role in the treatment of a variety of human solid tumors. However, its toxicity limits the clinical use. Recently, the administration of antioxidants has been suggested to protect against cisplatin-induced nephrotoxicity. The present study was designed to estimate the antitumor activity of the licorice extract alone and in combination with cisplatin, and its protective potential against cisplatin-induced toxicity in a mouse xenograft model. The administration of the licorice extract significantly inhibited tumor growth in BALB/C mice inoculated with CT-26 colon cancer cells. The combination of the licorice extract and cisplatin diminished the therapeutic efficacy of cisplatin but promoted considerably antitumor activity of the licorice extract. In mice with cisplatin treatment for 15 d, the serum levels of blood urea nitrogen and creatinine remarkably were increased by kidney damage, and the serum alanine aminotransferase and aspartate aminotransferase levels were elevated by liver damage. The administration of the licorice extract plus cisplatin recovered these functional indices in the kidney and liver to almost the control levels. In addition, the administration of the licorice extract significantly reduced the cisplatin-induced oxidative stress. Taken together, the administration of the licorice extract inhibits the growth of mouse colon carcinoma without any adverse effects, and reduces the cisplatin-induced toxicity. Therefore, the licorice extract may be a candidate for an anticancer and chemopreventive agent. However, cancer patients with cisplatin therapy should avoid the supplementation of the licorice extract.
Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. In our preliminary studies, CK has shown to exhibit anti-hyperglycemic effect through its insulin-secreting action, similar to that of insulin secretagogue sulfonylureas. Metformin, a biguanide, improves insulin resistance by reducing gluconeogenesis and enhancing peripheral glucose uptake, promoting reduction of the plasma glucose level. The aim of this study was to compare the anti-diabetic effects of CK and metformin due to differences in their mechanisms of action and also to investigate whether treatment of CK and metformin in combination show synergistic or additive effects compared to each drug alone. Seven week-old male db/db mice were treated for 8 weeks. CK was given at a dose of 10 mg/kg, metformin at 150 mg/kg and the same dosage of each drug was applied to CK plus metformin combination group. Significant improvements were observed in plasma glucose and insulin levels, homeostasis model assessment-insulin resistance (HOMA-IR) index and in hematoxylin and eosin-stained liver tissues in combination group. Although further studies to elucidate the benefits of co-administration of CK and metformin are needed, our findings may provide basis to the discovery of a new combination therapy on diabetes control in type 2 diabetics.
The antiallergic effects of magnolol and honokiol, isolated from the bark of Magnolia obovata (family Magnoliaceae), were investigated both in vitro and in vivo. Magnolol and honokiol potently inhibited passive cutaneous anaphylaxis reactions in mice induced by IgE–antigen complex as well as compound 48/80-induced scratching behaviors. These constituents exhibited not only potent inhibitory activity on the degranulation of RBL-2H3 cells induced by IgE–antigen complex, with IC50 values of 45 and 55 μM, respectively, but also inhibited the protein expressions of IL-4 and TNF-α. Based on these findings, magnolol and honokiol may improve IgE-induced allergic diseases.
During the course of experiments on the transformation of lignans to phytoestrogenic substances, such as enterodiol (END) and enterolactone (ENL), a previously isolated bacterium, Eubacterium (E.) sp. strain SDG-2, capable of phenolic p-dehydroxylation in the biotransformation of secoisolariciresinol diglucoside to END and ENL, was concluded to be Eggerthella (Eg.) lenta (Eg. sp. SDG-2) on the basis of 16S rRNA gene sequence analysis. The bacterium could transform (+)-dihydroxyenterodiol (DHEND, 3a) to (+)-END (1a), but not for (−)-DHEND (3b) to (−)-END (1b) under anaerobic conditions. By incubation of a mixture of (+)- and (−)-dihydroxyenterolactone (DHENL, 4a and 4b) with Eg. sp. SDG-2, only (−)-DHENL (4b) was converted to (−)-ENL (2b), selectively. On the other hand, we isolated a different bacterium, strain ARC-1, capable of dehydroxylating (−)-DHEND (3b) to (−)-END (1b) from human feces. Strain ARC-1 could transform not only (−)-DHEND (3b) to (−)-END (1b), but also (+)-DHENL (4a) to (+)-ENL (2b). However, the bacterium could not transform (+)-DHEND (3a) and (−)-DHENL (4b). Both bacterial strains demonstrated different enantioselective dehydroxylation.
It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCζ/λ). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.
The backbone structure of ginsenosides, active ingredients of Panax ginseng, is similar with that of sterol, especially cholesterol. Caenorhabditis elegans (C. elegans) is one of free living nematodes and is well-established animal model for biochemical and genetic studies. C. elegans cannot synthesize de novo cholesterol, although cholesterol is essential requirement for its growth and development. In the present study, we investigated the effects of ginseng total saponins (GTS) on the average brood size, growth, development, worm size, and life span of C. elegans in cholesterol-deprived and -fed medium. Cholesterol deprivation caused damages on normal growth, reproduction, and life span of worms throughout F1 to F3 generations. GTS supplement to cholesterol-deprived medium restored the growth, reproduction, and life span of worms as much as cholesterol alone-fed medium. GTS co-supplement to cholesterol-fed medium not only promoted worm reproduction but also induced bigger worms and faster growth than cholesterol-fed medium. In study to identify which ginsenosides are responsible for life span restoring effects of GTS, we found that ginsenoside Rc supplement not only restored life span of worms grown in cholesterol-deprived medium but also prolonged life span of worms grown in cholesterol-fed medium. Worms grown in medium supplemented with ginsenoside Rb1 or Rc to cholesterol-deprived medium exhibited strong filipin staining, in which filipin forms tight and specific complexes with 3β-hydroxy sterols. These results show a possibility that ginsenosides could be utilized by C. elegans as a sterol substitute and further indicate that ginsenoside Rc is the component of Panax ginseng that prolongs the life span of C. elegans.
In the course of our experiments on the metabolic conversion of lignans to the estrogenic substances enterodiol (END) and enterolactone (ENL) by human intestinal flora, we isolated two anaerobes, Ruminococcus sp. END-1 and strain END-2, capable of oxidizing END. The former selectively converted (−)-END to (−)-ENL, while the latter selectively converted (+)-END to (+)-ENL, indicating enantioselective oxidation by intestinal bacteria.
Nine anthraquinones, aurantio-obtusin (1), chryso-obtusin (2), obtusin (3), chryso-obtusin-2-O-β-D-glucoside (4), physcion (5), emodin (6), chrysophanol (7), obtusifolin (8), and obtusifolin-2-O-β-D-glucoside (9), isolated from an EtOAc-soluble extract of the seeds of Cassia tora, were subjected to in vitro bioassays to evaluate their inhibitory activity against advanced glycation end products (AGEs) formation and rat lens aldose reductase (RLAR). Among the isolates, compounds 6 and 8 exhibited a significant inhibitory activity on AGEs formation with observed IC50 values of 118 and 28.9 μM, respectively, in an AGEs-bovine serum albumin (BSA) assay by specific fluorescence. Furthermore, compounds 6 and 8 inhibited AGEs-BSA formation more effectively than aminoguanidine, an AGEs inhibitor, by indirect AGEs-ELISA. Nε-Carboxymethyllysine (CML)-BSA formation was also inhibited by compounds 6 and 8. Whereas compounds 1, 4, and 6 showed a significant inhibitory activity on RLAR with IC50 values of 13.6, 8.8, and 15.9 μM, respectively.
Most of the drug fraction penetrating the skin after topical application is taken up by the cutaneous blood flow, although the rest directly migrates into deeper tissues such as the subcutis and muscle. A new in situ experimental hairless rat model was designed to distinguish these fractions of topically applied drugs. Flurbiprofen, a non-steroidal anti-inflammatory drug, was selected as the model drug. In this model, two agar gel discs were subcutaneously inserted into the abdominal region of hairless rats as a drug receptor, and a topical formulation containing the drug was placed above either side of the gel disc. Plasma and agar levels of flurbiprofen were followed every 2 h over 10 h. The migration fraction of the drug into the systemic circulation and that directly to subcutaneous tissues were calculated to be 99.8% and 0.2% against the total amount which penetrated the skin, and the drug ratios into agar gel from the systemic circulation and not from the systemic circulation (i.e. directly migrated from the formulation) were 16.0% and 84.0%, respectively, at 10 h. This in situ drug disposition profile in skin was similar to the in vivo profile calculated from the in vivo muscle amount of flurbiprofen using muscle clearance. These results clearly suggest that the present in situ experimental model is a valuable tool for easy analysis of the skin disposition of topically applied drugs.
This study was carried out to investigate the in vitro effects of isopanduratin A and 4-hydroxypanduratin A isolated from Kaempferia pandurata ROXB. on melanin biosynthesis and tyrosinase activity. Two chalcone compounds, isopanduratin A and 4-hydroxypanduratin A, were isolated from the ethyl acetate fraction of ethanol extract as the active principles. Compared with phenylthiourea (IC50=34.3 μM) as a positive control, the depigmentation IC50 values for isopanduratin A and 4-hydroxypanduratin A were 10.6 μM and 23.2 μM, respectively. The compounds also significantly inhibited the activity of tyrosinase, the enzyme that converts DOPA (3,4-dihydroxyphenylalanine) to dopachrome in the biosynthetic process of melanin. The IC50 values of isopanduratin A and 4-hydroxypanduratin A for tyrosinase were 10.5 μM and >30 μM, respectively, while that of phenylthiourea was 47.6 μM. The tyrosinase protein level was also significantly decreased by isopanduratin A and 4-hydroxypanduratin A. The results indicate that isopanduratin A and 4-hydroxypanduratin A isolated from K. pandurata ROXB. are promising compounds that could be useful for treating hyperpigmentation as skin-whitening agents.
Carvedilol, an α- and β-adrenergic blocking drug, is mainly metabolized by CYP2D6, UGT1A1, UGT2B4 and UGT2B7. This drug is administered orally as a racemic mixture of R(+)- and S(−)-enantiomers. It has been reported that CYP2D6 prefers metabolizing S-carvedilol to R-carvedilol stereoselectively. On the other hand, stereoselective metabolism of carvedilol by UGTs is still unclear. Moreover, we have reported that patients with chronic heart failure who had polymorphism in CYP2D6, UGT1A1 and/or UGT2B7 had lower metabolic activity and oral clearance than did patients with no polymorphism. The aim of this study was to clarify stereoselective metabolism of carvedilol by UGT1A1 and UGT2B7 and to determine by using a recombinant enzyme-introduced mutation whether genetic mutation in UGT1A1 and UGT2B7 causes reduction in metabolic activity for carvedilol. A glucuronidation assay using human liver microsomes and recombinant UGT1A1 and UGT2B7 expressed in HeLa cells demonstrated that UGT1A1 prefers metabolizing R-carvedilol to S-carvedilol. On the other hand, UGT2B7 prefers metabolizing S-carvedilol to R-carvedilol. Moreover, G71R mutation of UGT1A1 reduced both affinity and capacity but did not affect stereoselective metabolism. On the other hand, both A71S and H268Y mutations of UGT2B7 reduced capacity but did not affect affinity and, as a result, the efficiency of metabolism was remarkably reduced. However, as in the case of UGT1A1, neither of the mutations affected stereoselective metabolism.
The population pharmacokinetic parameters of aripiprazole in healthy Japanese males were estimated using a nonlinear mixed effects model (NONMEM) program. Pharmacokinetic data for population analysis were obtained from the single-dose (24 subjects), multiple-dose (15 subjects), and itraconazole-coadministration (27 subjects) trials. The time course of plasma aripiprazole concentration following oral administration was well described by a two-compartment model with first-order input. The mean values of the absorption lag time (ALAG) and absorption rate constant (KA) were estimated to be 0.805 h and 2.65 h−1, respectively. The mean volume of the central (V1/F) and peripheral (V2/F) compartment was 3.84 and 1.54 l/kg, respectively, and the mean value of inter-compartment clearance (Q/F) was 0.168 l/h/kg. Oral clearance (CL/F) was estimated to be 0.0645 l/h/kg in the group with CYP2D6*1/*1, *1/*2 and *2/*2. The decrease in CL/F was estimated to be 0.0135 l/h/kg in the group with CYP2D6*1/*5, *1/*10, *2/*5, *2/*10, and *2/*41, and 0.0293 l/h/kg in the group with CYP2D6*5/*10, *10/*10, and *41/*41. The plasma concentration of aripiprazole was increased by coadministration of itraconazole, and the decrease in CL/F was estimated to be 0.0181 l/h/kg.
We performed a simulation for the clinical pharmacokinetic study, in which blood was sampled at two time points corresponding to the peak concentration (Cpeak) and trough concentration (Ctrough) following repetitive oral drug administration to subjects. We estimated the approximate oral clearance (CL/Fapprox) as 2·D/(Cpeak·τ+Ctrough·τ), where D is the dose, and τ is the dosing interval. The CL/Fapprox value was accurate for drugs with a long-elimination half-life, and the estimation error of the CL/F value was slightly increased for drugs with a shorter elimination half-life. The accuracy of CL/Fapprox in each subject was not affected by the magnitude of the interindividual pharmacokinetic variability, but was significantly decreased by the larger measurement error of drug concentrations (or intraindividual pharmacokinetic variability). We further performed several computer simulations to mimic statistical hypothesis testing following the clinical repeated-dose pharmacokinetic trials. The statistical power to detect the difference of oral clearance between two groups was marginally dependent on the measurement error of drug concentration, but was highly dependent on the interindividual pharmacokinetic variability. These findings suggested that the peak-and-trough sampling design to estimate the CL/Fapprox value is useful for clinical repeated-dose pharmacokinetic trials, and that the study design and protocol should be evaluated carefully by computer simulation prior to a real clinical trial.
We investigated the effect of Lactobacillus acidophilus strain L-55 isolated from infant feces on experimental allergic rhinitis in BALB/c mice. The heat-treated cells of strain L-55 were orally administrated for 4 consecutive weeks to mice sensitized by ovalbumin (OVA), and nasal symptoms (sneezing and nasal rubbing) induced by OVA challenge were evaluated. Strain L-55 at doses of 1 and 10 mg cells/mouse significantly inhibited nasal symptoms by repeated administration over a period of 2 weeks. Furthermore, we measured the level of OVA-specific IgE titers in the serum by passive cutaneous anaphylaxis (PCA) reaction. PCA titers in the sera from mice administrated strain L-55 were significantly lowered compared with the control. These results suggest that oral administration of strain L-55 may be useful for alleviating the nasal symptoms of allergic rhinitis.
To characterize the membrane transport responsible for the renal excretion and intestinal absorption of levo-floxacin, we performed pharmacokinetic analysis of transcellular transport across LLC-PK1 and Caco-2 cell monolayers. Transcellular transport of levofloxacin in LLC-PK1 cells was greater in the basolateral-to-apical direction than in the opposite direction. Pharmacokinetic analysis indicated that basolateral uptake was the direction-determining step for the transcellular transport of levofloxacin in LLC-PK1 cells. The apical efflux clearance of levofloxacin in LLC-PK1 cells was increased at the medium pH 6 as compared with at pH 8, suggesting that membrane transport characteristics of levofloxacin are apparently similar to those of a prototypical organic cation, tetraethylammonium. On the other hand, transcellular transport of levofloxacin in Caco-2 cells was only slightly greater in the basolateral-to-apical direction than in the opposite direction. The apical efflux clearance of levofloxacin in Caco-2 cells was greater than basolateral efflux clearance, and apical influx clearance was greater than any other membrane transport clearance. In addition, the apical uptake of levofloxacin as well as quinidine in Caco-2 cells was inhibited significantly by nicotine and imipramine. The findings indicated that some transporters are responsible not only for the efflux but also for the influx of levofloxacin at the apical membrane of Caco-2 cells.
The purpose of this study was to evaluate the physicochemical properties, skin permeation and accumulation profiles of model lipophilic ketorolac fatty ester (esters) prodrugs. Ketorolac linoleate (C18:2), oleate (C18:1) and stearate (C18:0) were evaluated for their solubility, capacity factor, enzymatic hydrolysis, chemical stability, and skin permeation and accumulation profiles using the combination of common permeation enhancing techniques such as the use of supersaturated solution of permeants in the enhancer vehicle, lipophilic receptor solution, enhancer pretreatment of skins, removal of stratum corneum and delipidization of skins etc. Esters were highly lipophilic, chemically stable for the duration of observation, enzymatically unstable in hairless mouse skin/liver homogenates and plasma, and impermeable into the receptor solution. Absence of skin permeation, relative enzymatic stability during permeation and chemical stability of these esters could delineate preliminary possibilities for designing safer topical agents without systemic absorption.
The last of four histamine receptors (HRs), H4 receptor (H4R), was identified in the year 2000. Since that time, the H4R has been implicated in cellular mechanisms related to immune systems, inflammatory processes, and allergic reactions. We have previously reported the expression of H4R mRNA in rheumatoid arthritic (RA) synovial cell cultures by means of the RT-PCR method. The present study demonstrates the expression of H4R at the protein level in RA synovial cell cultures. We also identified that the receptor was expressed in two specific types of cell populations, fibroblast-like and macrophage-like cells from RA synovial tissues. In the culture system, the fifth generation of fibroblast-like synoviocytes showed as strong expression of H4R as in the primary culture. The results provide pragmatic evidence for H4R as a novel target in the development of pharmacotherapeutic agents for RA treatment. Furthermore, the culture system used here will be useful for future studies concerning basic molecular mechanisms underlying the pharmacology of H4R.
Plasma concentration profiles of arundic acid ((R)-(−)-2-propyloctanoic acid), an oil-like medicine, administered as soft-gel capsules in human clinical tests were predicted from the dissolution test data of the soft-gel capsules with different storage terms (short- and long-term stored drugs) by applying the in vitro–in vivo correlation (IVIVC). We established two linear-regression IVIVCs, which were characterized by either the in vitro dissolution behaviors against the pH 8.0 dissolution medium or the pH 6.8 dissolution medium containing 2% sodium dodecyl sulfate (SDS), in this study. Also, the prediction accuracies for the in vivo plasma profiles in humans for these two IVIVCs were compared. Regarding dissolution from the long-term stored capsule in pH 8.0 dissolution medium without surfactant, the prediction accuracies of the in vivo plasma profiles in humans were not satisfactory for the obtained IVIVC. The use of pH 6.8 dissolution medium containing 2% SDS, according to the Japanese guideline, improved the dissolution of the long-term stored capsule. Furthermore, the prediction accuracies for the in vivo plasma profiles in humans for these two IVIVCs were compared. The IVIVC established by the in vitro dissolution data obtained with the dissolution medium containing surfactant more effectively predicted the plasma drug concentration profiles following oral administrations of the soft-gel capsules under both storage conditions.
Compound injections of oxycodone and hydrocotarnine are currently used as one of the treatment options for some cases with cancer pain. However, there have been no reports examining the factors that influence oxycodone and hydrocotarnine clearance, so detailed examination is necessary. As for hydrocotarnine, there have been no reports examining the pharmacokinetics. Therefore in this study, we determined the pharmacokinetics of oxycodone and hydrocotarnine in patients with cancer pain. The study was conducted on 19 patients, in whom pain control was attempted by using the compound injections of oxycodone and hydrocotarnine. We used HPLC-electrochemical detector (ECD) to determine oxycodone and hydrocotarnine serum concentrations, and used the nonlinear least-squares method (MULTI) for calculation of the pharmacokinetic parameters. Furthermore, we examined the factors that influence the clearance of oxycodone and hydrocotarnine by multiple regression analysis (step wise method). The pharmacokinetic parameters were as follows: Oxycodone; Vd=226.7±105.5 l (mean±S.D.), CL=37.9±25.1 l/h, t1/2=4.1±1.9 h. Hydrocotarnine; Vd=276.8±237.2 l, CL=95.1±64.3 l/h, t1/2=2.0±0.7 h. The clearance of oxycodone represented by a regression formula was significantly correlated to the age, the presence or absence of within 7 d on the death or liver metastasis, or of the heart failure of the patients. The clearance of hydrocotarnine represented by a regression formula was significantly correlated to the presence or absence of within 7 d on the death or liver metastasis, or of the heart failure of the patients. The clearance also indicated that oxycodone concentration in the blood was likely to be higher in patients having these factors. Oxycodone/hydrocotarnine compound injections should be used with caution and dose reduction may be necessary in such populations.
To establish a strategy for developing 111In-diethylenetriaminepentaacetic acid (111In-DTPA)-octreotide, a diagnostic radiopharmaceutical agent for tumors, with reduced non-specific renal radio-accumulation, the compounds having D-glutamic acid (Glu) or γ-carboxy-D-glutamic acid (carboxy-Glu) as the N-terminal amino acid were examined for in vivo radio-distribution. Compounds carrying Glu and carboxy-Glu containing one and two negative charges, respectively, showed lower renal radio-accumulation than that carrying D-phenylalanine. It was revealed that the introduction of a negative charge reduces the renal radio-accumulation independently from the number of negative charges. The present result can be a clue for the development of 111In-DTPA-octreotides with reduced the renal radio-accumulation.
We have developed a novel method for the identification of Curcuma longa and C. aromatica called “loop-mediated isothermal amplification (LAMP),” based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5—10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.