Construction of integrated system for selection of fused plant protoplasts

Summary An integrated system has been constructed to instantly identify and efficiently sort the heterokaryons formed by plant protoplast fusion. The system is composed of the following functions: a) a transport system, b) an electro-manipulator, c) a cell harvester, d) a flow cytometer/cell sorter, and e) a control device. The conditions for an efficient and reproducible enrichment of the heterokaryons have been investigated by this system using the fluorescein isothiocyanate stained protoplasts preparing from Glycyrrhiza glabra cell cultures and unstained protoplasts of Abrus precatorius cell cultures which contain a large quantity of chlorophyll.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscissic acid - FITC fluorescein isothiocyanate This paper is part 96 in the series Studies on Plant Tissue Cultures. For part 95 see Orinara Y., Noguchi T. and Furuya T. (1993) submitted for publication.     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca²⁺-activated Cl⁻ current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron-like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108-15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans - d,l -1-amino-1,3-cyclopentanedicarboxylate on the neuron-like P19 cells induced intracellular Ca²⁺ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR-induced phenomena at the molecular level.     

Localization of the antigen for the monoclonal antibody ②9F11-B-E4 interspecific cross-reaction in the freshwater triclads

The localization of the antigen for monoclonal antibody 9F11-B-E₄ was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed.     

In vitro study on release of thyroid hormone in solitary autonomously functioning thyroid nodules using cell culture method

In vitro release of thyroid hormone was investigated under basal and TSH-stimulated conditions in the solitary autonomously functioning thyroid nodules (AFTN). A small portion (0.5 g of wet weight) of the nodules and adjacent thyroid tissues removed surgically from five patients with solitary AFTN were prepared for the dispersed cell culture. In the experiment on non TSH-stimulated (basal) conditions, those culture media which were totally replaced on the 5th day after primary culture were utilized for the determination of thyroxine (T4) and triiodothyronine (T3) by radioimmunoassay. T4 and T3 levels in culture media of the functioning nodules were 1.15 +/- 0.33 microgram/dl (mean +/- SEM) and 2.72 +/- 0.68 ng/ml, contrasted with levels of 0.67 +/- 0.09 microgram/dl and 1.24 +/- 0.22 ng/ml in the paranodular tissues. The mean ratios of T3/T4 of the nodules and paranodular tissues were 0.25 +/- 0.02 and 0.19 +/- 0.02, respectively (p less than 0.05). Meanwhile, in another experiment under TSH stimulatory conditions employing 40 and 80 microU/ml of human TSH, there were no significant differences in T4 and T3 releases when the two groups were compared.     

Presence of adrenocorticotropin-potentiating factors in porcine thyroid glands

An extract of porcine thyroid gland in 0.1 N acetic acid exerted dose-dependent potentiation of ACTH-induced corticosterone production in isolated rat adrenal cells. The extract by itself manifested no steroidogenic activity. Upon gel-filtration of the extract, potentiating activities were demonstrated in three main peaks with molecular weights of about 10,000, 5,000 and 2,000. These findings indicate the presence of heterogeneous forms of ACTH-potentiating factors in the thyroid. Significant enhancement of ACTH-induced steroidogenesis was readily apparent with three gel-filtration fractions at a lower concentration of ACTH (4.75 pM). At this concentration, dose-dependent potentiation was observed with these three fractions. Enhanced corticosterone production responses by cells preincubated with the thyroid extract were observed and the results indicated the existence of potentiating mechanisms other than inhibition of ACTH proteolysis. The lack of T4, T3 and thyroglobulin in this activity suggests that the activity resides in other constituents of the thyroid.     

Sulphides and furanones from steam volatile oils of Allium fistulosum and Allium chinense

The constituents of the steam volatile oils from two kinds of Allium fistulosum, A. fistulosum var. caespitosum and A. chinense, have been investigated by GC and spectral techniques (IR, UV, GC/MS, ¹H NMR and ¹³C NMR). The compounds identified from the neutral fraction of each volatile oil included sulphides, thiolanes, alcohols, aldehydes, ketones, furanones and others. Among the sulphur compounds, dipropyl disulphide comprised ca 28% of A. fistulosum oil, ca 23% of A. fistulosum var. caespitosum oil and ca 30% of A. chinense oil. A. fistulosum oil was characterized by a large quantity of tridecan-2-one (ca 52%) and 2,3-dihydro-2-octyl-5-methylfuran-3-one (ca 16%). Also, a large amount of 2,3-dihydro-2-hexyl-5-methylfuran-3-one (ca 20%) was isolated from A. chinense oil.     

Complement-dependent cytotoxicity of sera obtained from subacute sclerosing panencephalitis patients

Human lymphoid cells (NC-37) persistently infected with either measles virus (Schwarz and TYCSA strains) or subacute sclerosing panencephalitis (SSPE) virus (Halle and Mantooth strains) were destroyed in the presence of complement by anti-measles sera as well as by sera from SSPE patients. The cytotoxic activity was demonstrated in both IgG and IgM fractions of measles convalescent sera, but only in IgG fraction of SSPE sera. Measles convalescent sera completely lost the cytotoxic activity to all the cell lines, when absorbed with any one of the cell lines, indicating that the viral surface antigens of these cell lines infected with measles or SSPE virus are identical. On the other hand, the cytotoxic activity of SSPE sera could not be readily absorbed with these cells. Thus, the affinity of SSPE sera for the viral surface antigens might be lower than that of measles convalescent sera.