Establishment and characterization of human uterine cervical epidermoid carcinoma cell line HHUS containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.     

Cloning of a<Emphasis Type="Italic"> decapentaplegic</Emphasis> orthologue from the sawfly,<Emphasis Type="Italic"> Athalia rosae</Emphasis> (Hymenoptera), and its expression in the embryonic appendages

The cDNA of a decapentaplegic (dpp) orthologue from the sawfly, Athalia rosae (Hymenoptera), was cloned and characterized. The clone (Ar dpp) was 2,566 bp long and encoded 395 amino acids in a single open reading frame. Genomic Southern blotting showed that Ar dpp is a single copy gene. The deduced amino acid sequence can be aligned along its entire length with known insect DPPs. It shared common characteristics such as a signal sequence, a pro-domain region, and a ligand domain with seven cysteines at conserved locations. Ar dpp was expressed as a single 5.0-kb mRNA in embryos, larvae, pupae and adults. In situ hybridization showed that Ar dpp was expressed in the dorsal region proper in early embryonic stages and in the embryonic appendages of cephalic segments (labrum, antenna, mandible, maxilla, and labium), thoracic segments (thoracic legs), and all abdominal segments except the tenth segment (pleuropodia and proleg primordia). The present results indicate that Ar dpp expression reflects the primary determination of embryonic appendages.Edited by D. TautzThe sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB121072     

Phase 1 study of multiple biomarkers for metabolism and oxidative stress after one-week intake of broccoli sprouts

Little is known about the direct effect of broccoli sprouts on human health. So we investigated the effect of broccoli sprouts on the induction of various biochemical oxidative stress markers. Twelve healthy subjects (6 males and 6 females) consumed fresh broccoli sprouts (100 g/day) for 1 week for a phase 1 study. Before and after the treatment, biochemical examination was conducted and natural killer cell activity, plasma amino acids, plasma PCOOH (phosphatidylcholine hydroperoxide), the serum coenzyme Q(10), urinary 8-isoprostane, and urinary 8-OHdG (8-hydroxydeoxyguanosine) were measured. With treatment, total cholesterol and LDL cholesterol decreased, and HDL cholesterol increased significantly. Plasma cystine decreased significantly. All subjects showed reduced PCOOH, 8-isoprostane and 8-OHdG, and increased CoQ(10)H(2)/CoQ(10) ratio. Only one week intake of broccoli sprouts improved cholesterol metabolism and decreased oxidative stress markers.     

Phytochemical intake and relationship to past health history in Japanese women

We calculated functional food factor (FFF) intakes using a new database and examined their relationship to health conditions commonly affecting Japanese women in midlife. One-day DRs were collected weekly for 6 months from 67 Japanese women, aged 45-55 yr, living in Kyoto prefecture, Japan. Macro- and micronutrient and FFF intake were calculated from the resulting 1528 DRs. Factor analysis and logistic regression were performed to identify relationships between FFFs and past health history. Fourteen of 17 FFF factors, as well as age, BMI and menopausal status, exhibited both positive and negative correlations with past history of hypertension, diabetes, allergy, migraine, and menopausal syndrome.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

The pheromone production of female Plodia interpunctella is inhibited by tyraminergic antagonists

Several compounds were found to suppress the calling behavior and in vitro pheromone biosynthesis of the Indian meal moth, Plodia interpunctella. The compounds were screened by means of a calling-behavior bioassay with female P. interpunctella. Five derivatives with activities in the nanomolar range were identified, in order of decreasing pheromonostatic activity: 4-hydroxybenzaldehyde semicarbazone (42) > 5-(4-methoxyphenyl)-1,3-oxazole (38) > 5-[4-(tert-butyl)phenyl]-1,3-oxazole (40) > 5-(3-methoxyphenyl)-1,3-oxazole (35) > 5-(4-cyanophenyl)-1,3-oxazole (36). These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis, as determined with isolated pheromone-gland preparations that incorporated [1-(14)C]sodium acetate in the presence of the so-called pheromone-biosynthesis-activating neuropeptide (PBAN). The non-additive effect of the inhibitor with antagonist (yohimbine) for the tyramine (TA) receptor suggests that it could be a tyraminergic antagonist. Three-dimensional (3D) computer models were built from a set of compounds. Among the common-featured models generated by the program Catalyst/HipHop, aromatic-ring (AR) and H-bond-acceptor-lipophilic (HBAl) features were considered to be essential for inhibitory activity in the calling behavior and in vitro pheromone biosynthesis. Active compounds, including yohimbine, mapped well onto all the AR and HBAl features of the hypothesis. Less-active compounds were shown to be unable to achieve an energetically favorable conformation, consistent with our 3D common-feature pharmacophore models. The present hypothesis demonstrates that calling behavior and PBAN-stimulated incorporation of radioactivity are inhibited by tyraminergic antagonists.     

Ingestion of proanthocyanidins derived from cacao inhibits diabetes-induced cataract formation in rats

Proanthocyanidins derived from cacao (CLP) have various antipathophysiological functions. We have tested whether dietary supplementation with CLP prevents cataract formation in rats with diabetes induced by streptozotocin (STZ), using histological, histochemical, and biochemical analyses. Starting at 7 days after the streptozotocin challenge, the animals were fed either a normal diet or a diet containing 0.5% w/w CLP over 10 weeks. There were no significant differences in plasma and urine glucose concentrations, plasma fructose amines, and plasma thiobarbituric reactive substances (TBARS) between the two dietary groups. Antioxidant status as assessed by measuring lipid peroxide production in plasma in response to azocompounds was lower in the STZ-rats fed control diet than in animals fed CLP. Opacity was first detected in the lenses of the control dietary group 5 weeks after STZ injection and cataracts had developed in the majority of these animals by 10 weeks. These changes were rarely seen in the STZ/CLP diet group. Histological examinations of the eyes of the STZ-treated normal diet group revealed focal hyperplasia of the lens epithelium and liquefaction of cortical fibers. There were similar but considerably less severe changes in the animals fed CLP. Hydroxynonenal (HNE), a marker of oxidative stress, was detected immunohistochemically in the lenses of the STZ-treated normal diet group, but not of those receiving CLP. Our findings suggest that CLP inhibits diabetes-induced cataract formation possibly by virtue of its antioxidative activity.     

Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.     

Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study

The aim of the present study was to investigate the efficiencies of producing transgenic rats by the ooplasmic injection of sperm heads (intracytoplasmic sperm injection: ICSI) and elongating spermatids (elongating spermatid injection: ELSI) exposed to the EGFP DNA solution. A slightly lower proportion of ICSI oocytes using sperm heads exposed to a concentration of 0.5 microg/ml DNA solution for 1 min developed into offspring (13.3%, 48/361) when compared to that of oocytes injected with nontreated sperm heads (19.4%, 32/165). Eight ICSI offspring were found to be EGFP-carrying transgenic rats (16.7% per offspring; 2.2% per embryo). After a 1-min exposure of the elongating spermatids to 5 microg/ml of DNA solution, 8.8% (45/511) of the ELSI oocytes developed into offspring while 12.7% (22/173) of the ELSI oocytes using nontreated spermatids developed. Six ELSI offspring carried the EGFP DNA (13.3% per offspring; 1.2% per embryo). The conventional pronuclear microinjection of 5 microg/ml of DNA solution resulted in the higher production of offspring (29.7%, 104/350) and the birth of three transgenic rats (2.9% per offspring; 0.9% per embryo). Thus, sperm heads and elongating spermatids were practically useful as the vector of exogenous DNA if the DNA-exposed spermatogenic cells were microinseminated into rat oocytes.     

Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders

Glycosphingolipid (GSL) lysosomal storage disorders are a small but challenging group of human diseases to treat. Although these disorders appear to be monogenic in origin, where the catalytic activity of enzymes in GSL catabolism is impaired, the clinical presentation and severity of disease are heterogeneous. Present attitudes to treatment demand individual therapeutics designed to match the specific disease-related gene defect; this is an acceptable approach for those diseases with high frequency, but it lacks viability for extremely rare conditions. An alternative therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to balance cellular GSL biosynthesis with the impairment in catalytic activity seen in lysosomal storage disorders. The development of N-alkylated iminosugars that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase, offers a generic therapeutic for the treatment of all glucosphingolipidoses. The successful use of N-alkylated iminosugars to establish SRT as an alternative therapeutic strategy has been demonstrated in in vitro, in vivo and in clinical trials for type 1 Gaucher disease. The implications of these studies and the prospects of improvement to the design of iminosugar compounds for treating Gaucher and other GSL lysosomal storage disorders will be discussed.