Cleft lip in oculodentodigital dysplasia suggests novel roles for connexin43.

Oculodentodigital Dysplasia (ODDD) is a rare syndrome involving anomalies in eye, tooth, and digit formation, caused by mutations in CX43/GJA1. In addition to classic dental features, ODDD includes oral and craniofacial accessory symptoms such as characteristic facial appearance and cleft palate. However, there have been no reports of ODDD accompanied by cleft lip. Herein we report, for the first time, a male, sporadic, Asian proband presenting bilateral cleft lip. By direct sequence analysis, our proband was diagnosed as having ODDD with a heterozygous mutation, codon 142 G>A in GJA1 and CX43E48K. We excluded the possibility of pathogenic mutations in B3GALTL, BMP4, TFAP2A, PVRL1, IRF6, and MSX1. To address how CX43/GJA1 is related to cleft lip, we performed immunohistochemistry using mouse and human mid-facial tissue. CX43 expression was detected in the nasal compartment and nasal and maxillary processes at murine developmental stage E12.5. Furthermore, CX43 expression was found in the epithelial tissue inside the human subepithelial cleft lip that completes epithelial fusion. Therefore, we suggest that CX43/GJA1 is involved in lip formation. Our case report of ODDD with a bilateral cleft lip suggests that CX43/GJA1 might be a novel candidate gene for syndromic cleft lip.

[Left ventricular pacemaker-lead implantation through left thoracotomy for cardiac resynchronization therapy].

We performed direct implantation of a left ventricular pacemaker-lead through left thoracotomy for cardiac resynchronization therapy (CRT). We exposed the left ventricular free wall using a pericardium lifting method without hemodynamic deterioration or arrhythmia. Intraoperative ultrasonic cardiography is useful for determining a suitable implantation site for CRT.

Detection of antibodies to Japanese encephalitis virus in the wild boars in Hiroshima prefecture, Japan.

Serum specimens were collected from 25 wild boars in Hiroshima prefecture located in the western region of Japan from November 2004 to February 2005. The sera were tested for antibodies to Japanese encephalitis virus (JEV) by IgM capture and IgG enzyme-linked immunosorbent assays (ELISA), and plaque reduction neutralization test. Seventeen samples (68%) were positive for neutralizing antibody to JEV. All the neutralizing antibody-positive samples were positive for IgG-ELISA. One was also positive for IgM. The results indicate that approximately 70% of the wild boars were positive for anti-JEV antibody, and raises the possibility that wild boars may play a role in the infectious cycle of JEV in this region.

Cancer chemoprevention by ginseng in mouse liver and other organs.

Oral administration of red ginseng extracts (1% in diet for 40 weeks) resulted in the significant suppression of spontaneous liver tumor formation in C3H/He male mice. Average number of tumors per mouse in control group was 1.06, while that in red ginseng extracts-treated group was 0.33 (p<0.05). Incidence of liver tumor development was also lower in red ginseng extracts-treated group, although the difference from control group was not statistically significant. Anti-carcinogenic activity of white ginseng extracts, besides red ginseng extracts, was also investigated. In the present study, the administration of white ginseng extracts was proven to suppress tumor promoter-induced phenomena in vitro and in vivo. It is of interest that oral administration of the extracts of Ren-Shen-Yang- Rong-Tang, a white ginseng-containing Chinese medicinal prescription, resulted in the suppression of skin tumor promotion by 12-o-tetradecanoylphorbol-13-acetate in 7,12-dimethylbenz[a]anthracene-initiated CD-1 mice. These results suggest the usefulness of ginseng in the field of cancer prevention.

Direct injection method for quantitation of endogenous leukotriene E4 in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry with a column-switching technique.

A new analytical method employing liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a column-switching system was developed for quantitative determination of leukotriene E4 (LTE4) in human urine. A column-switching system using a trapping column, which concentrates the analyte and removes salts and other water-soluble contaminants, allowed direct injection of human urine. Because simultaneously eluted endogenous contaminants suppressed the ionization efficiency of LTE4, good liquid chromatographic separation was very important for establishing this method, notwithstanding the high selectivity of MS/MS. The calibration curve was linear over the range from 10 to 3000 pg/mL, and the method showed good accuracy and precision. This method should therefore be very useful for determination of LTE4 amounts in human urine in studies on leukotriene metabolism and the efficacy of antileukotriene drugs.

Oxidation mechanism of 7-hydroxy-delta 8-tetrahydrocannabinol and 8-hydroxy-delta 9-tetrahydrocannabinol to the corresponding ketones by CYP3A11.

A cDNA isolated from a C57BL/6 mouse liver cDNA library had the identical nucleotide sequence in coding region with the mouse CYP3A11, and the NH(2)-terminal sequence was also identical to that of cytochrome P450 (P450) MDX-B, a microsomal alcohol oxygenase. The COS-7 cells transfected with the CYP3A11 expression vector formed 7-oxo-Delta(8)-tetrahydrocannabinol (7-oxo-Delta(8)-THC) from 7alpha- and 7beta-hydroxy-Delta(8)-THC. An immunologically related protein with P450 MDX-B was expressed in the COS-7 cell microsomes. The cell microsomes expressed CYP3A11; COS-3A11 catalyzed the oxidation of 7-hydroxy-Delta(8)-THC and 8-hydroxy-Delta(9)-THC to 7-oxo-Delta(8)-THC and 8-oxo-Delta(9)-THC, respectively, in a reconstituted system. (18)O derived from atmospheric oxygen was incorporated into about 30% of the corresponding ketones formed from 7alpha-hydroxy-Delta(8)-THC and 8beta-hydroxy-Delta(9)-THC by mouse hepatic microsomes, P450 MDX-B, and COS-3A11, although incorporation of the stable isotope into the oxidized metabolites from 7beta-hydroxy-Delta(8)-THC and 8alpha-hydroxy-Delta(9)-THC was negligible. (18)O, however, was not incorporated into 7-oxo-Delta(8)-THC formed from 7alpha-hydroxy-Delta(8)-THC by using cumene hydroperoxide instead of NADPH under (18)O(2). When (18)O-labeled 7alpha-hydroxy-Delta(8)-THC and 8beta-hydroxy-Delta(9)-THC were incubated with above enzymes under air, about 30% of the ketones formed released (18)O from a hydroxy group at the 7 and 8 positions in the course of the oxidation. These results suggest that 7alpha-hydroxy-Delta(8)-THC and 8beta-hydroxy-Delta(9)-THC may be oxidized to the corresponding ketones by CYP3A11 via a gem-diol pathway. 7beta-Hydroxy-Delta(8)-THC and 8alpha-hydroxy-Delta(9)-THC may be also converted to the ketones through a stereoselective dehydration of an enzyme-bound gem-diol rather than through a direct hydrogen extraction as a peroxy form of the enzyme.

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

TMC-69, a new antitumor antibiotic with Cdc25A inhibitory activity, produced by Chrysosporium sp. TC1068. Taxonomy, fermentation and biological activities.

A new antibiotic designated TMC-69 has been isolated from the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69 exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69 prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25A and B phosphatases.

Murine homologs of deltex define a novel gene family involved in vertebrate Notch signaling and neurogenesis.

Notch signaling plays an important role in cell-fate specification in multicellular organisms by regulating cell-cell communication. The Drosophila deltex gene encodes a modulator of the Notch pathway that has been shown to interact physically with the Ankyrin repeats of Notch. We isolated four distinct cDNAs corresponding to mouse homologs of deltex - mouse Deltex1 (MDTX1), mouse Deltex2 (MDTX2), mouse Deltex2DeltaE (MDTX2DeltaE), and mouse Deltex3 (MDTX3). Deduced amino acid sequences of these four cDNAs showed a high degree of similarity to Drosophila Deltex and its human homolog, DTX1 throughout their lengths, even though they possess distinct structural features. MDTX proteins formed homotypic and heterotypic multimers. We found that these genes were expressed in the central, peripheral nervous system and in the thymus, overlapping with those of mouse Notch1. In mammalian tissue culture cells, overexpression of any of the four mouse deltex homologs suppressed the transcriptional activity of E47, a basic helix-loop-helix (bHLH) protein, in a manner similar to suppression by an activated form of human Notch1 or human DTX1. In addition, overexpression of MDTX2 and MDTX2DeltaE in C2C12 cells under differentiation-inducing conditions suppressed the expression of myogenin, one of the myogenic transcriptional factors; this was also similar to a previously reported activity of constitutively activated Notch. Furthermore, misexpression of any of the MDTX genes in Xenopus embryos resulted in an expansion of the region expressing the neural cell adhesion molecule (N-CAM) gene, a marker for the neuroepithelium. Collectively, our results suggest that these mouse deltex homologs are involved in vertebrate Notch signaling and regulation of neurogenesis.

CYP3A4 is a major isoform responsible for oxidation of 7-hydroxy-Delta(8)-tetrahydrocannabinol to 7-oxo-delta(8)-tetrahydrocannabinol in human liver microsomes.

The human liver enzyme microsomal alcohol oxygenase was able to oxidize both 7alpha- and 7beta-hydroxy-Delta(8)-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-Delta(8)-THC) to 7-oxo-Delta(8)-THC. The oxidative activity was determined by using a panel of 12 individual cDNA-expressed human cytochrome P450s (CYPs) (1A1, 1A2, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Met, 2D6-Val, 2E1 and 3A4). Among the CYP isoforms examined, CYP3A4 showed the highest activity for both of substrates. The metabolism of 7alpha- and 7beta-hydroxy-Delta(8)-THC to 7-oxo-Delta(8)-THC was also detected for CYPs 1A1 (4.8% of CYP3A4), 1A2 (4.7%), 2A6 (2.3%), 2C8 (16.6%), and 2C9-Cys (5.4%), and CYPs 1A1 (0.4%), 2C8 (1.3%), 2C9-Arg (4.3%), and 2C9-Cys (0.9%), respectively. The 7alpha- and 7beta-hydroxy-Delta(8)-THC microsomal alcohol oxygenase activities in human liver were significantly inhibited by addition of 100 microM troleandomycin, 1 microM ketoconazole, and anti-CYP3A antibody, although these activities were not inhibited by 1 microM 7, 8-benzoflavone and 50 microM sulfaphenazole. When the substrates were incubated with the CYP3A4-expressed microsomes under oxygen-18 gas phase, atmospheric oxygen was incorporated into 35% of 7-oxo-Delta(8)-THC formed from 7alpha-OH-Delta(8)-THC, but only 12% of 7-oxo-Delta(8)-THC formed from 7beta-OH-Delta(8)-THC. These results indicate that CYP3A4 is a major isoform responsible for the oxidation of 7alpha- and 7beta-hydroxy-Delta(8)-THC to 7-oxo-Delta(8)-THC in liver microsomes of humans, although the oxidation mechanisms for 7alpha- and 7beta-hydroxy-Delta(8)-THC might be different.

Saponins isolated from Allium chinense G. Don and antitumor-promoting activities of isoliquiritigenin and laxogenin from the same drug.

Investigation of the Chinese crude drug "Xiebai," the bulbs of Allium chinense G. Don (Liliaceae), led to the isolation of 2 saponins, xiebai-saponin I (laxogenin 3-O-beta-xylopyranosyl (1-->4)-[alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside) (1) and laxogenin 3-O-alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside (2), and the aglycone, laxogenin (3), together with 2 chalcones, isoliquiritigenin (4) and isoliquiritigenin-4-O-glucoside (5), and beta-sitosterol glucoside (6). Compounds 1-5 were tested in vitro for their inhibitory effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of HeLa cells. In addition to this, laxogenin (3) was proven to have an antitumor-promoting activity in a two-stage lung carcinogenesis experiment.

TMC-171A,B,C and TMC-154, novel polyketide antibiotics produced by Gliocladium sp. TC 1304 and TC 1282.

Four new antibiotics, TMC-171A (2), B (3), C (4) and TMC-154 (5) have been isolated from the fermentation of fungal strains Gliocladium sp. TC 1304 and TC 1282, respectively. Spectroscopic and degradation studies have shown that TMC-171s and TMC-154 were new members of the TMC-151 class of antibiotics, unique polyketides modified with a D-mannose and a D-mannitol or a D-arabitol. These compounds showed moderate cytotoxicity to various tumor cell lines.

Major cytochrome P450 enzyme responsible for oxidation of secondary alcohols to the corresponding ketones in mouse hepatic microsomes. Oxidation of 7-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol.

The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-Delta8-THC) to 7-oxo-Delta8-THC in hepatic microsomes of mice were significantly increased by the treatment of mice with dexamethasone or phenobarbital. A cytochrome P450 enzyme, named P450MDX-B, was purified from hepatic microsomes of dexamethasone-treated mice, and its apparent molecular mass was estimated to be 51,000. The NH2-terminal amino acid sequence of P450MDX-B was the same as that of CYP3A11. The oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC were 2.55 and 4.92 nmol/min/nmol P450, respectively. The antibody against P450MDX-B almost completely inhibited the oxidative activities of 7alpha- and 7beta-hydroxy-Delta8-THC in mice. These results indicate that P450MDX-B (CYP3A11) is a major enzyme responsible for the oxidation of 7alpha- and 7beta-hydroxy-Delta8-THC to 7-oxo-Delta8-THC in mouse liver.

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer.

BACKGROUND & AIMS: Microsatellite instability (replication error [RER]) is a characteristic of tumors in hereditary nonpolyposis colon cancer (HNPCC), but the mechanism of HNPCC carcinogenesis is not yet understood. To clarify the nature of HNPCC tumors, RER and genetic changes were compared between HNPCC and non-HNPCC tumors. METHODS: RER and genetic changes were analyzed in 21 HNPCC, 389 familial adenomatous polyposis, and 206 sporadic tumors using polymerase chain reaction, single-strand conformation polymorphism, sequencing, and Southern hybridization. RESULTS. in HNPCC, 95% tumors at all stages showed RER positivity (altered loci, 4.3 of 5). In familial adenomatous polyposis and sporadic tumors, RER positivity (1.7 of 5) was 3% in adenoma and intramucosal carcinoma, 13%-24% in invasive carcinoma, and 35% in carcinoma metastasized to liver. Fifty percent of RER-positive HNPCC tumors had both germline and somatic mutations of hMSH2 or hMLH1 gene, whereas 6% of RER-positive non-HNPCC had somatic mutation. APC, p53, and K-ras-2 mutations and loss of heterozygosity of tumor-suppressor genes were significantly less frequent (P = 0.03 to 0.0006) but transforming growth factor beta type II receptor mutation was significantly more frequent (P = 0.000001) in HNPCC than in non-HNPCC. CONCLUSIONS: RER positivity occurs from an early stage of carcinogenesis in HNPCC but in later stages in non-HNPCC. Most HNPCC tumors may develop through different genetic changes from those in the adenoma-carcinoma sequence, although a certain percentage develops through APC mutation.

Tissue distribution of macromolecular conjugate, adriamycin linked to oxidized dextran, in rat and mouse bearing tumor cells.

Tissue distribution of the radioactivities after intravenous administration of [14C]adriamycin ([14C]ADM) or [14C]ADM linked to oxidized dextran ([14C]ADM-OXD) in mouse bearing Lewis lung carcinoma (LLC) and rat bearing Walker 256 carcinosarcoma was studied. ADM conjugated with OXD increased plasma half-life and gave high area under the plasma concentration-time curve (AUC). The AUC values were 13.0 and 5.8 times higher than those of the [14C]ADM group in mice and rats, respectively. In the tumor tissues, AUC values of the [14C]ADM-OXD group were also respectively 1.6 and 1.9 times higher than those of the [14C]ADM group. However, the AUC values in the heart of the [14C]ADM-OXD group were about half those of [14C]ADM group in both animals. Thus the distribution of ADM was changed by the conjugation with OXD. The excretion profile of ADM was also changed by the conjugation. During 6 h after administration, [14C]ADM-OXD was mainly excreted into rat urine at 45.2% of the original dose, but in the [14C]ADM group recovery in urinary excretion was 4.2%. Using [14C]ADM-OXD and ADM-[14C]OXD, the respective tissue distribution of ADM and OXD portions in the ADM-OXD was studied in rats bearing Walker 256. The radioactivities of both [14C]ADM-OXD and ADM-[14C]OXD groups increased in tumor and liver within 1 h after administration. In the liver, both radioactivities were retained for 24 h, which suggested that ADM and OXD were retained as conjugated form, however, different behavior was observed between the two groups in tumor tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane.

Endothelin-1 (ET-1), a 21 amino-acid potent vasoconstrictor peptide, is produced from the biologically inactive intermediate big ET-1 via an endoproteolytic cleavage between Trp-21 and Val-22 by endothelin converting enzyme (ECE). cDNA sequence analysis predicts that the two other members of the endothelin family, ET-2 and ET-3, are also generated from the corresponding intermediates called big ET-2 and big ET-3, respectively. The metalloproteinase inhibitor phosphoramidon inhibited the conversion of big ET-1 into mature ET-1 both in vivo and in cultured endothelial cells, suggesting that ECE may be a neutral metalloproteinase. In this study, we solubilized and partially purified ECE from the membrane fraction of porcine lung. Using gel filtration chromatography, we separated two distinct ECE activities, designated M1 (apparent molecular mass approx. 300 kDa) and M2 (approx. 65 kDa). Optimum pH for the cleavage of big ET-1 by M1 and M2 was 7.0 and 7.5, respectively. M1 efficiently converted human big ET-1(1-38) to ET-1, but not human big ET-2(1-37) or human big ET-3(1-41)-amide. In contrast, M2 converted both big ET-1 and big ET-2, but not big ET-3. M1 was inhibited by phosphoramidon (IC50 approx. 1 microM) but not by thiorphan or bacitracin. In contrast, M2 was inhibited by much lower concentrations of phosphoramidon (IC50 approx. 0.3 nM), as well as by thiorphan and bacitracin. ECE activity in M1 was able to bind to a concanavalin A-agarose column and was eluted by alpha-methyl-D-glucoside, indicating that the ECE is glycosylated. From these results, M1 and M2 from the porcine lung membrane are similar to the candidate of ECE in endothelial cells and neutral endopeptidase in kidney (EC 3.4.24.11), respectively. Taken in conjunction with the previous finding that neither thiorphan nor bacitracin affected the conversion of endogenously synthesized big ET-1 in cultured endothelial cells, we conclude that physiologically relevant ECE found in the endothelial cells is more similar to M1 than to M2.

Agrimoniin, an antitumor tannin of Agrimonia pilosa Ledeb., induces interleukin-1.

The induction of interleukin-1 (IL-1) by agrimoniin, a tannin of Agrimonia pilosa Ledeb., in human peripheral blood mononuclear cells (PBMC) in vitro and in mouse adherent peritoneal exudate cells (PEC) in vivo was studied. A significant amount of IL-1 beta in the culture supernatant of the human PBMC stimulated with agrimoniin was detected with an enzyme-linked immunoadherent assay. Agrimoniin induced IL-1 beta secretion dose- and time-dependently. The adherent PEC from mice intraperitoneally injected with agrimoniin (10 mg/kg) also secreted IL-1 4 days later. These results suggest that agrimoniin, a plant tannin, is a novel cytokine inducer.