Anti-platelet effects of chalcones from Angelica keiskei Koidzumi (Ashitaba) in vivo.

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine that is also regarded in Japan as a health food with potential antithrombotic properties. The ability of the major chalcones, xanthoangelol (XA) and 4-hydroxyderricin (4-HD) extracted from Ashitaba roots to inhibit platelet aggregation activity in vitro was recently determined. However, the anti-platelet activities of Ashitaba chalcones in vivo have remained unclear. The present study examines the anti-platelet effects of Ashitaba exudate and its constituent chalcones using mouse tail-bleeding models that reflect platelet aggregation in vivo. Ashitaba exudate and the major chalcone subtype XA, suppressed the lipopolysaccharide (LPS)-induced shortening of mouse tail bleeding. However, trace amounts of other Ashitaba chalcone subtypes including xanthoangelols B (XB), D (XD), E (XE) and F (XF) did not affect tail bleeding. These results suggest that the major chalcone subtype in Ashitaba, XA, has anti-platelet-activities in vivo.

Should isolated peritoneal carcinomatosis from colorectal cancer be sub-classified into stage IVB in era of modern chemotherapy?

BACKGROUND: According to the 7th edition of the TNM staging system, stage IV metastatic colorectal cancer (CRC) at the time of initial diagnosis is sub-classified into stage IVA or IVB disease. Peritoneal carcinomatosis (PC), considered to have a dismal prognosis, is exclusively sub-classified into stage IVB, even though other metastases to a sole organ are sub-classified into stage IVA, which is considered to be associated with better survival. This retrospective study was undertaken to investigate the overall survival in metastatic CRC patients, focusing on PC patients. METHODS: We reviewed data on patients with metastatic CRC at initial diagnosis surgically treated between January 2006 and June 2011. A survival analysis was performed paying special attention to PC and sub-classifying patients with PC into three categories according to metastatic sites. RESULTS: There were 69 stage IVA patients (IVA group) and 83 stage IVB. Among stage IVB patients, 20 had isolated PC (PC-I group), 28 had PC with one or more other sites of metastasis (PC-II group), and 35 had at least 2 metastatic without peritoneal involvement (NPC group). Of 152 stage IV patients, 132 (87 %) underwent resection of the primary tumor and 19 (12 %) underwent radical resection of metastatic disease with microscopic free margins (R0 resection) including 5/20 (25 %) patients in the PC1 group. A total of 139 patients received oxaliplatin-based chemotherapy in a palliative (n = 125), neoadjuvant (n = 3), or adjuvant setting after R0 resection (n = 11). Compared with 36.6 months in the PC-I group, median survival was 32.5 months (P = 0.48) in the IVA group, 14.7 months (P = 0.07) in the PC-II group, and 12.9 months (P < 0.01) in the NPC group. CONCLUSIONS: The sub-classification of isolated PC into stage IVA instead of IVB might be more appropriate in the era of modern chemotherapy. Further investigation is warranted.

A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart.

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope.

We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels.

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.

Decreases in cAMP phosphodiesterase activity in hepatocytes cultured with herbimycin A due to cellular microtubule polymerization related to inhibition of tyrosine phosphorylation of alpha-tubulin.

The increase in cellular cAMP concentration during 10-min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine-phosphorylation of cellular proteins including alpha-tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin-induced tyrosine phosphorylation, and overcome by the addition to the 10-min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and 4-(3-butyloxy-4-methoxyphenyl)-2-imidazolidinone (Ro-20-1724, a PDE4 inhibitor), in addition to 3-isobutyl-1-methylxanthine (a nonselective inhibitor). Rapid breakdown of the once-accumulated cAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 microM during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule-disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine-containing medium was further supplemented with taxol, a microtubule-stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol-like and vinblastine-suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein-bound anti-(alpha-tubulin). Thus, 4-h culture of hepatocytes with herbimycin inhibits phosphorylation of the C-terminal tyrosine residue of alpha-tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism.

BAZF, a novel Bcl6 homolog, functions as a transcriptional repressor.

The BCL6 gene, which has been identified from the chromosomal translocation breakpoint in B-cell lymphomas, functions as a sequence-specific transcriptional repressor. We cloned a novel Bcl6-homologous gene, BAZF (encoding Bcl6-associated zinc finger protein). The predicted amino acid sequence of BAZF indicated that the BTB/POZ domain and the five repeats of the Krüppel-like zinc finger motif are located in the NH2-terminal region and the COOH-terminal region, respectively. BAZF associated with Bcl6 at the BTB/POZ domain and localized in the nucleus. Since zinc finger motifs of BAZF were 94% identical to those of Bcl6 at the amino acid level, BAZF bound specifically to the DNA-binding sequence of Bcl6 and functioned as a transcriptional repressor. The repressor activity was associated with both the BTB/POZ domain and the middle portion of BAZF. The 17-amino-acid sequence in the middle portion was completely conserved between BAZF and Bcl6, and the conserved region was critical for the repressor activity. Expression of BAZF mRNA, like that of Bcl6 mRNA, was induced in activated lymphocytes as an immediate-early gene. Therefore, the biochemical character of BAZF is similar to that of Bcl6 although the tissue expression pattern of BAZF differs from that of Bcl6. This is apparently the first report of a gene family whose members encode zinc finger proteins with the BTB/POZ domain.

Mercury-sensitive residues and pore site in AQP3 water channel.

Water channel function of all aquaporins (AQPs) but AQP4 can be inhibited by mercurial reagents. Mercurial reagents are believed to bind specifically to cysteine residues and block the aqueous pore of AQPs. Because of the low homology of AQP3 to other AQPs, it is not certain whether the pore structure of AQP3 is similar to that of the others. Determination of mercury-sensitive cysteine residues in AQP3 and comparison with those in other AQPs will help to resolve this question. When AQP3 was expressed in Xenopus oocytes, incubation with 0.3 mM HgCl2 decreased its osmotic water permability (Pf) by approximately 30%. To identify the mercury-sensitive site, six individual cysteine residues in human AQP3 (at positions 11, 29, 40, 91, 174, and 267) were altered by site-directed mutagenesis. Mutants of C11S and C11A had a similar basal Pf to wild-type but acquired mercury resistance. Replacement of Cys-11 with Trp, which possesses a large side chain, did not change Pf. Mercurial inhibition of Pf was still observed in five other Cys-to-Ser mutants. These results suggest that Cys-11 is the mercury-sensitive residue in AQP3 and that this residue might be independent of water channel function. Mutation of Tyr-212, a position corresponding to the mercury-sensitive residues in AQP1 and AQP2, to cysteine enhanced the mercurial inhibition of Pf. Y212W had no water channel activity. Expression of AQP3 increased glycerol permeability (Pgly) 3.1-fold, whereas Pgly of Y212W-expressing oocytes was similar to Pgly of control oocytes. Cysteine mutation at Tyr-212 increased the inhibitory effect of mercury on Pgly. These results suggest that the structure of the aqueous pore of AQP3 resembles those of AQP1 and AQP2 and support the hypothesis that water and small molecules share a common pore in AQP3.

[Neoadjuvant intra-arterial chemotherapy based on chemosensitivity tests for locally invasive bladder cancer].

We investigated the clinical usefulness of individualization of chemotherapeutic regimen in neoadjuvant intra-arterial chemotherapy for locally invasive bladder cancer. Anticancer drugs were selected according to the results of an in vitro chemosensitivity test (collagen matrix assay or succinic dehydrogenase inhibition test). Nine patients with locally invasive bladder cancer received 1 to 4 courses of neoadjuvant intra-arterial chemotherapy, followed by radical cystectomy. Histopathological responses in the cystectomized specimens were grade 3 in 3 cases, grade 2 in 2, grade 1b in 2 and no response in 2. Pathologically, a complete response and downstaging were observed in 3 and 4 cases, respectively. Seven of the 9 patients were alive no evidence of disease with a mean follow-up period of 38.9 months, whereas 2 patients died of metastasis within 2 years. Six of the 7 patients who showed a complete response or down staging have been free of recurrence. These findings suggest that our chemotherapeutic strategy may improve the prognosis for locally invasive bladder cancer.

Surface electromyographic electrode pair with built-in buffer-amplifiers.

By means of a surface electrode with an operational amplifier, a new electrode unit suitable for an electromyographic-biofeedback apparatus and for portable electromyography used outside a Faraday cage was developed. The operational amplifier, which has an output impedance lower than 10 ohms, functions as an efficient buffer amplifier and is able to protect the EMG signals from background noises. This new electrode unit is small (32 x 12 x 5 mm), waterproof, and inexpensive. Because its structure is simple, it can be built in any laboratory.

Disposition of 6-chloro-2-pyridylmethyl nitrate, a new anti-anginal compound, in rats and dogs.

1. The absorption, distribution, metabolism and excretion of 6-chloro-2- pyridylmethyl nitrate, a new anti-anginal compound, were investigated in rats and dogs after intravenous and peroral administration of the 14C-labelled or unlabelled drug. 2. The half-lives of plasma levels for the alpha and beta phase and systemic availability were 6 min, 42 min and 26-50% respectively in rats, and 8 min, 66 min and 5% respectively in dogs. 3. Radioactivity was rapidly distributed in the tissues of rats, and recovered mainly in the 0-24 h urine (95% of dose within 24 h) with no excretion in the expired air. 4. Several metabolites were detected on t.l.c. of rat and dog urine, and four were identified as N-(chloro-2-pyridylcarbonyl)-glycine (M1, 56%), N-acetyl-S-(6- chloro-2-pyridylmethyl)-L-cysteine (M2, 29%), 6-chloro-2-pyridinecarboxylic acid (M3, 5%) and 6-chloro-2-pyridylmethyl. beta-D-glucuronate (M4, 7%). No unchanged drug was excreted.