Chemical constituents of Millettia taiwaniana: structure elucidation of five new isoflavonoids and their cancer chemopreventive activity

We describe the isolation and identification of five new isoflavonoids, millewanins A (1), B (2), C (3), D (4), and E (5), together with six known isoflavonoids and three rotenoids, from the stems of Millettia taiwaniana collected in Japan. The major component, auriculasin (6), exhibited significant inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. The results of the present investigation indicate that 6 might be a valuable antitumor promoter.     

Changes in serum tumor necrosis factor (TNF-alpha) with kami-shoyo-san administration in depressed climacteric patients

An herbal medicine (kampo) is widely used to prevent or treat climacteric symptoms. In order to investigate the potential involvement of tumor necrosis factor (TNF)-alpha in susceptibility to mood disorder in climacteric women and to clarify the relationship between immune function and the efficacy of herbal medicine, we compared serum TNF-alpha levels in two treated groups, with and without concurrent use of herbal medicine. This study included 113 consecutive depressed menopausal patients who visited the gynecological and psychosomatic medicine outpatient clinic of the Osaka Medical College Hospital in Japan. Fifty-eight patients were administered kami-shoyo-san according to the definition of above sho. In contrast, 55 patients who were different in sho of kami-shoyo-san were administered antidepressants. Hamilton Rating Scale for depression (HAM-D) scores were determined at baseline and 12 weeks after starting treatment (endpoint). TNF-alpha concentrations were analyzed before and after 12 weeks of treatment. Kami-shoyo-san significantly increased plasma concentrations of TNF-alpha after 12 weeks of treatment, to 17.22 +/- 6.13 pg/ml from a baseline level of 14.16 +/- 6.27 pg/ml (p = 0.048). The percent change in plasma concentration of TNF-alpha differed significantly between the kami-shoyo-san therapy group and the antidepressant therapy group at 4 weeks (12.0 +/- 7.8% and -1.22 +/- 0.25%, respectively, p < 0.01), 8 weeks (19.7 +/- 3.4% and -2.45 +/- 0.86%, respectively, p < 0.01), and 12 weeks (21.3 +/- 5.4% and -6.81 +/- 2.2%, respectively, p < 0.001). We found in this study that kami-shoyo-san, an herbal medicine, increased plasma TNF-alpha levels in depressed menopausal patients. Cytokines may play various roles in mood and emotional status via the central nervous system and may be regulated by herbal medicines, although the interactions are very complex.     

ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC

BACKGROUND: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP-binding Walker's motif As, and mutations in these regions affect the clock oscillations. RESULTS: ATP induced the hexamerization of KaiC. The Km value for the ATP for the hexamerization was 1.9 micro m. Triphosphate nucleotides bound to the two Walker's motif As, and their binding functioned cooperatively for the hexamerization. An unhydrolysable substrate, 5'-adenylylimidodiphosphate (AMPPNP), also induced the hexamerization, indicating that nucleotide binding, but not its hydrolysis, is essential for the hexamerization. Mutations in each of the two Walker's motif As that affect the clock phenotype increased the Km value for ATP and inhibited the hexamerization. Thus, the KaiC hexamerization seems to be necessary for its clock function. The KaiC hexamer has the shape of a hexagonal pot with a diameter and height of approximately 100 A and with a relatively large cavity (73 A deep and 18-34 A wide) inside. This pot-shaped structure suggests that KaiC functions in a similar manner to F1-ATPase, helicase or ATP-dependent protease/chaperon, all of which have dynamic activities inside the central cavity of their hexameric rings. CONCLUSION: ATP-induced KaiC hexamerization is necessary for the clock function of KaiC.     

Growth arrest of PC12 cells by nerve growth factor is dependent on the phosphatidylinositol 3-kinase/Akt pathway via p75 neurotrophin receptor

We recently isolated mutant PC12 cell clones (PC84 cells) by transfection of PC12 cells with nerve growth factor (NGF) cDNA. These cells secreted active NGF and extended short processes, but proliferated faster than the parental PC12 cells. Because the expression level of p75, a low-affinity receptor for NGF, was significantly low, we suspected that NGF signaling via p75 was necessary for the growth arrest of the PC12 cells, and this was shown to be the case by repressing p75 function in PC12 cells. In this study, we examined the downstream signaling of p75, which would ultimately evoke the growth arrest. NGF is known to induce rapid phosphorylation of MAP kinase and Akt in PC12 cells, whereas in PC84 cells, MAP kinase was phosphorylated but the phosphorylation level of Akt was very low under the serum-free condition. This finding suggested that the low expression level of p75 in PC84 cells was the reason for the low Akt activation. Because Akt is known to be activated via phosphatidylinositol (PI) 3-kinase, we treated PC12 cells with a PI3-kinase inhibitor, Wortmannin, and found these cells did not cease proliferation in the presence of NGF. Furthermore, anti-p75 neutralizing antibody reduced NGF-induced phosphorylation of Akt in PC12 cells under the serum-free condition. Because we had already shown that PC12 cells treated with anti-p75 neutralizing antibody did not cease proliferation in the presence of NGF, these results suggest that NGF activates Akt via p75, which is necessary for the NGF-induced growth arrest of PC12 cells.     

Reliability,validity and standardization of the Japanese version of the Social Adjustment Scale-Self Report

The purpose of the present paper was to examine the reliability and validity of the Japanese version of the Social Adjustment Scale-Self Report (SAS-SR) and to present its normative data. The SAS-SR was administered to a random sample of all the employees of a large general hospital, together with the General Health Questionnaire (n = 363). It was also administered to a representative subset of first-visit patients at 33 psychiatric hospitals and clinics from all over Japan, along with the semistructured psychiatric interview to ascertain the patients' diagnoses (n = 1581). For the internal consistency reliability of the subscales and the overall scale of the SAS-SR, Cronbach's alpha was between 0.61 and 0.73. The Pearson product-moment correlations between the subscale and overall scale scores with the GHQ score were mostly >0.3. The scores were statistically significantly and substantively different between the normal sample and the patient samples, and were also meaningful, differentiating between various diagnostic subgroups. The reference ranges of the SAS-SR scores for mentally healthy subjects were calculated as 95% prediction intervals; for example, 1.22-2.22 for the overall score. The Japanese version of the SAS-SR has good reliability and satisfactory validity. The present study provided reference ranges for its scores in order to increase their interpretability. With its ease of administration and its rich subscales, the scale promises to offer a psychometrically sound measure with which to assess social adjustment in people with various psychiatric disorders.     

Antro-pancreatic reflexes: long- and short-route reflexes in exocrine and endocrine pancreatic secretion in dogs

Pancreatic exocrine secretion is known to be facilitated by gastric antral distension via long- and short-route reflexes. In this study, we studied the effects of gastric distension on intra-pancreatic nerve discharges and blood insulin level as well as pancreatic exocrine secretion. Mongrel dogs were anesthetized with ketamine and thiopental, and immediately decerebrated. This study consisted of two series of experiments. In the first series, efferent discharges in an intra-pancreatic nerve branch were recorded, and its responses to antral distension were analyzed. In the second series, effects of antral distension on pancreatic exocrine secretion and blood insulin level were observed before and after vagotomy in splanchnicectomized dogs. Efferent discharges in a pancreatic nerve branch were increased by antral distension. Neither vagotomy nor splanchnicectomy produced obvious changes in the neural response. In splanchnicectomized dogs, antral distension elevated blood insulin level and increased pancreatic exocrine secretion. After subsequent vagotomy, these effects were reduced, but the increases were still greater than 50%. These results indicate that the antro-pancreatic short-route reflex plays a significant role in exocrine secretion, and also suggest that insulin release is increased by antral distension independent of blood glucose level.     

Binding site(s) on P-glycoprotein for a newly synthesized photoaffinity analog of agosterol A

Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.     

Various functions of ClC-type Cl- channels

Cellular functions of Cl- channels are poorly understood, in contrast to well-established roles of cation channels. Recently, important achievements in Cl- channel research have been sequentially reported, including cloning of many Cl- channel cDNAs, linkage of gene abnormalities to human inherited disorders, analysis of knock-out mouse phenotype, analysis of crystal structure, and regulation by protein-protein interaction. Intracellular membrane Cl- channels are important for acidification of intracellular vesicles: ClC-5 functions for re-absorption of low-molecular-weight proteins in renal proximal tubule, and ClC-7 for absorption of bone matrix by osteoclasts. Abnormal functions of these channels result in Dent's disease characterized by proteinuria and kidney stones and by osteopetrosis, respectively. Plasma membrane Cl- channels, ClC-K1, ClC-K2, and ClC-3B, are expressed predominantly in epithelial cells and are important for uni-directional Cl- transport across the epithelia. Abnormalities of these channels are also related to human diseases: abnormal ClC-K1 to diabetes insipidus and abnormal ClC-K2 to Bartter's syndrome.