A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question

For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.     

Cloning and expression of beta1,4-galactosyltransferase gene from Helicobacter pylori

Helicobacter pylori, which is a human pathogen associated with gastric and duodenal ulcer, has been shown to express human oncofetal antigens Lewis X and Lewis Y. Although the mammalian glycosyltransferases that synthesize these structures are well characterized, little is known about the corresponding bacterial enzymes. We report that a novel beta1,4-galactosyltransferase gene (HpgalT) involved in the biosynthesis of lipopolysaccharides in H. pylori has been cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein (HpGal-T) encoded by HpgalT consists of 274 residues with the calculated molecular mass of 31,731 Da, which does not show significant similarity to those of beta1,4-galactosyltransferases from mammalian sources and Neisseria It was confirmed that HpGal-T catalyzed the introduction of galactose from UDP-Gal in a beta1,4 linkage to accepting N-acetylglucosamine (GlcNAc) residues by means of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). When the E.coli cells which overexpressed HpgalT was coupled with the UDP-Gal production system, which consisted of recombinant E.coli cells overexpressing its UDP-Gal biosynthetic genes and Corynebacterium ammoniagenes, N-acetyllactosamine, a core structure of lipopolysaccharide of H.pylori, was efficiently produced from orotic acid, galactose, and GlcNAc.     

Molecular recognition of cAMP by an RNA aptamer

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.     

HMB-45 staining for cytology of primary melanoma of the vagina. A case report

BACKGROUND: Malignant melanoma in the vagina is very rare, but its diagnosis is usually easy if a melanin pigment is present. With cytodiagnosis, however, it is difficult to differentiate amelanotic melanoma or scantily pigmented melanoma from other conditions. In the present case, monoclonal antibody HMB-45, the efficacy of which has been established in histologic studies, was used in the cytodiagnosis of amelanotic melanoma in the vagina. CASE: A woman, aged 78 years, presented with a brownish, nodular tumor, diameter 3 cm, in the vagina. Scraping smears with Papanicolaou staining showed nonepithelial malignant cells without granules suggesting melanin. Smears stained with HMB-45 showed positive immunoreactivity. The diagnosis underwent histologic confirmation of amelanotic melanoma on the initial biopsy. CONCLUSION: Cytodiagnosis was made with HMB-45, which proved very effective in the differential cytodiagnosis of amelanotic melanoma and scantily pigmented melanoma, particularly because it obviated the need for tissue invasion.     

Biologically active oligodeoxyribonucleotides. Part 12: N2-methylation of 2'-deoxyguanosines enhances stability of parallel G-quadruplex and anti-HIV-1 activity

2'-Deoxyguanosine residues of a 3',5'-end-modified hexadeoxyribonucleotide (R-95288) with anti-HIV-1 activity were substituted with N2-methyl-2'-deoxyguanosine (m2dG). These modified oligodeoxyribonucleotides (ODNs) showed a 2-fold higher activity than R-95288. Also, the CD spectra of these ODNs indicated that the m2dG modification stabilized the tertiary structure of the G-quadruplex.     

Isolation and characterization of di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins) produced by reverse action of alpha-mannosidase from jack bean

Di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins, beta CDs), which were synthesized together with monomannosyl-beta CD in a large-scale production by reverse action of alpha-mannosidase from jack bean, were isolated and purified by HPLC. The structures of seven isomers of di-mannosyl-beta CD and six isomers of tri-mannosyl-beta CD were elucidated by FABMS and NMR spectroscopy, and by enzymatic methods.     

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.     

Evaluation of two-dimensional electronic portal imaging device using integrated images during volumetric modulated arc therapy for prostate cancer

BackgroundThe aim of the study was to evaluate analysis criteria for the identification of the presence of rectal gas during volumetric modulated arc therapy (VMAT) for prostate cancer patients by using electronic portal imaging device (EPID)-based in vivo dosimetry (IVD).Materials and methodsAll measurements were performed by determining the cumulative EPID images in an integrated acquisition mode and analyzed using PerFRACTION commercial software. Systematic setup errors were simulated by moving the anthropomorphic phantom in each translational and rotational direction. The inhomogeneity regions were also simulated by the I’mRT phantom attached to the Quasar phantom. The presence of small and large air cavities (12 and 48 cm³) was controlled by moving the Quasar phantom in several timings during VMAT. Sixteen prostate cancer patients received EPID-based IVD during VMAT.ResultsIn the phantom study, no systematic setup error was detected in the range that can happen in clinical (< 5-mm and < 3 degree). The pass rate of 2% dose difference (DD2%) in small and large air cavities was 98.74% and 79.05%, respectively, in the appearance of the air cavity after irradiation three quarter times. In the clinical study, some fractions caused a sharp decline in the DD2% pass rate. The proportion for DD2% < 90% was 13.4% of all fractions. Rectal gas was confirmed in 11.0% of fractions by acquiring kilo-voltage X-ray images after the treatment.ConclusionsOur results suggest that analysis criteria of 2% dose difference in EPID-based IVD was a suitable method for identification of rectal gas during VMAT for prostate cancer patients.