Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.     

High-performance Liquid Chromatography of Aflatoxins with Fluorescence Detection

Aflatoxins B_l, B₂, G₁ and G₂ were quantitatively detected by high-performance liquid chromatography on a 12 µl flow-cell in the fluorometric detector using the mobile phase of toluene system instead of chloroform, dichloromethane or methanol system. Various kinds of columns and mobile phases were tested, and fine mutual separation of all the four aflatoxins without quenching their fluorescence was achieved by using sHica gel column and toluene- ethyl acetate-formic acid-methanol (89.0: 7.5: 2.0: 1.5 v/v/v/v). The relationship between the fluorescence peak area and the amount injected was linear in the range of 0.3 ng to 120 ng. This method, as applied to food and feed extracts, is sensitive at the 10~20 ppb levels of the four kinds of aflatoxins.     

Effect of Validamycin on Production of Some Enzymes in Rhizoctonia solani

A remarkable effect of validamycin on the morphology of Rhizoctonia solani was seen after 2 days culture when the fungus was cultivated in a Roux flask with standing. In accordance with the morphological change, the production of laminarinase and glucan synthetase by the fungus was affected by validamycin.

The production of laminarinase was increased in the culture filtrate, and significantly decreased in the mycelium in the presence of validamycin. While the intracellular production of glucan synthetase in the culture with validamycin (10~50μg/ml) increased by 40~60% compared with that in the control culture.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix.

Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH(2) terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.     

Significant interaction between low-spin iron(III) site and pyrroloquinoline quinone in active center of nitrile hydratase

Nitrile hydratase, a unique non-heme iron enzyme, contains low-spin Fe(III) site and pyrroloquinoline quinone in the active center. The enzymatic activity of nitrile hydratase is strongly inhibited by typical carbonyl reagents such as phenylhydrazine and semicarbazide. Of special interest is the fact that these carbonyl reagents induced significant alteration of the ESR features of the native iron(III) enzyme at pH 7.2. In addition, the isobutyronitrile(inhibitor)-bound enzyme was also remarkably affected by phenylhydrazine and then the transformed ESR characteristics were different from those of phenylhydrazine-treated enzyme. The present results reveal the first evidence for an important interaction between the low-spin Fe(III) site and pyrroloquinoline quinone which is essential for the activity of nitrile hydratase.     

Separation of mammalian cell surface proteins by a two-dimensional gel electrophoresis system

Separation of externally exposed plasma membrane proteins of mammalian cells has been achieved by a new two-dimensional gel electrophoresis system. The proteins were separated in the first dimension on cylindrical polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) and in the second dimension on polyacrylamide slab gels containing 9 M urea, 0.1% SDS, and 0.1% Triton CF10. Using this method we have obtained reproducible high-resolution patterns of cell surface proteins of differentiated rat neuro-tumor cells in culture and of normal rat retinal cells. Different cell types show characteristic cell surface proteins in addition to ubiquitous ones. The number of common surface proteins between two cell types account for approximately half of the total surface proteins. By immunoprecipitation we have also found that rabbit anti-serum against a rat neuronal cell line can recognize most of these external proteins. Since the separation in the first dimension is done in the presence of SDS and the second dimension in the presence of SDS, a non-ionic detergent, and urea, the technique is particularly suitable for proteins that are of poor solubility. In addition to size, net charge and hydrophobicity appear to be important factors in the separation. Virtually all of the proteins that run in the first dimension can be recovered and further separated in the second.     

The Alarm Pheromone of the Mite Suidasia medanensis OUDEMANS, 1924 (Acariformes,Suidasiidae)

The stored-product mite Suidasia medanensis has been found in human-provided habitats in Japan. The mite uses neral as the alarm pheromone, and also secretes (Z,E)- and (£,£)-farnesal, which were isolated from an astigmatid mite for the first time. On the other hand, S. medanensis is the only mite out of fifteen species investigated so far that does not secrete volatile hydrocarbons along with the alarm pheromone, though 9-nonadecene and 6,9-nonadecadiene were found in a hexane extract of the mite. GC-MS, ^JH-NMR and ¹³C-NMR of the four isomers of synthetic farnesal, which were isolated by LC and preparative GLC, cleared up some confusion in the literature concerning the stereochemistry of this sesquiterpenoid.