Five Candidate Genes for Hamster Cardiomyopathy Did not Map to the Cardiomyopathy Locus by FISH Analysis

The Syrian cardiomyopathic hamster (BIO14.6), that developsboth muscular dystrophy and progressive cardiomyopathy, is widelyused as an animal model of autosomal recessive cardiomyopathymimicking human hypertrophic cardiomyopathy, and five geneshave been proposed as strong candidates for the cause of cardiomyopathy.We recently mapped the cardiomyopathy locus of the hamster tothe centromeric region of chromosome 9qa2.1-b1 by constructionof a genetic linkage map of the Syrian hamster. Thus, we analyzedthe loci of the five candidate genes, tropomyosin, cardiactroponin T, adhalin, calpain 3 and cardiac myosin binding protein-C,by the FISH method, and found that these genes were mapped onthe distal portion of chromosome 12qa5 and 4pa2 and the proximalportion of chromosomes 9qb7, 1qc1.1 and 1qb3, respectively.These results provide strong evidence that the five candidategenes previously proposed are not related to the hamster cardiomyopathy.     

A single cell observation of staurosporine effect on the Ca signals in rat basophilic leukemia cells

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

The GTP-Binding Proteins, Go and Gi2, of Neural Cloned Cells and Their Changes During Differentiation

The levels of the alpha-subunits of two GTP-binding proteins, Go and Gi2, were determined in neural and nonneural cloned cells by immunoassays. Go alpha was detected in all neural cells and some of nonneural cells, but not in HL-60 leukemic cells and PYS-2 teratocarcinoma-derived cells. The level of Go alpha was highest in the PC12 pheochromocytoma cells. Gi2 alpha was present in all types of cells tested, and its level was highest in the HL-60 cells and relatively high in glioma cells. Treatment of PC12 cells and neuroblastoma x glioma hybrid NG108-15 cells with nerve growth factor and forskolin, respectively, caused the extension of neuronal-like processes and increase in the level of Go alpha by 60-80%, but small changes in the levels of Gi2 alpha.     

On the physiology and chemistry of fern antheridiogens

Döpp has demonstrated an antheridium-inducing hormone (antheridiogen) in P.aquilinum. This antheridiogen (abbr. Apt.) is active in many, if not all, species of the family Polypodiaceae. Among responsive species, the minimally effective concentration varies widely. Apt was assayed againstOnoclea sensibilis because this species fails to form antheridia spontaneously under the prevailing conditions of culture and because none of the many species tested responds to a lower concentration A_Pt is inactive toward the investigated species of the fern families Osmundaceae, Cyatheaceae and Schizaeaceae. The two schizaeaceous speciesAnema phyllitidis andLygodium japonicum also elaborate antheridiogens (abbr. A_An and A_Ly). Both these antheridiogens are inactive in 0.sensibilis, the species used to assay for A_Pt. A_An, A_Ly, A_Pt and A_On (the antheridiogen of O.sensibilis) are distinct entities based both on the criteria of cross-testing and of Chromatographic separation. Cross-testing led to the conclusion that the antheridiogen ofCeratopteris thalictroides differ from A_Pt and A_An. Gibberellins have antheridiogenic properties in schizaeaceous species but, like A_An and A_Ly, they fail to hasten antheridium formation in the species used to assay for A_Pt. The native antheridiogens of schizaeaceous species are more species-selective in their action than is GA₃. A_An has recently been isolated. Its structure is similar to, if not identical with, that of gibberellins. A_An behaves like a weak gibberellin in several higher plant assay systems. The prothalli ofP. aquilinum andO. sensibilis become insensitive to Apt as they attain heart shape or shortly thereafter. Prothalli ofP. aquilinum do not begin to synthesize A_Pt and secrete it into the medium, until after they have become insensitive to it. It is in consequence of this that the most rapidly growing and developing individuals attain the archegonial phase without a prior antheridial phase. Various mechanisms and developmental characteristics are described, which strongly favor cross-fertilization inP. aquilinum without, however, eliminating an opportunity for self-fertilization. The cells of abortive antheridium initials, and of “green antheridia”, exhibit certain characteristics of green vegetative cells. These atypical structures appear to arise when early antheridial stages are overtaken by conditions unfavorable to antheridium differentiation. The observations suggest that A_Pt may be required beyond an initial inductive event. The investigations led to the conclusion that A_Pt functions by cancelling a light-dependent block to antheridium formation and suggest that in darkness this block decays without the intervention of A_Pt. InPolypodium crassifolium, the light-effect on antheridium formation is mediated by phytochrome. Other subject matters discussed include: The cellular location of antheridium initials; the relationship of antheridiogen to antheridiogen structure; the existence of a switching mechanism in the sexual development ofO. sensibilis; the retrieval of genetic information in the induction and differentiation of antheridia; the tempero-spatial pattern of competence to antheridiogen in schizaeaceous species and the inducibility of a physiological state antagonistic to antheridium formation in A.phyllitidis.     

Mating Triggers Queen Elimination by Workers of Japanese Harvester Ant (<Emphasis Type="Italic">Messor aciculatus</Emphasis>)

During reproduction, ant colonies produce winged queens. These new queens usually leave the nest to mate and can then establish a new nest. If the new nest is close to an existing colony, it will be in competition with the existing colony. Therefore, workers will kill any mated queens they find outside the colony during the reproductive season. In this study, factors that might determine whether workers eliminate queens were investigated. Mating status (mated or unmated), colony origin (same or different to tested workers) and mating partners (inbred or outbred) of the queens of Japanese harvester ants (Messor aciculatus) were manipulated and the workers’ behavior towards the queens was observed. Mated queens were always attacked by workers, though this was not affected by either colony origin or mating partners. These results suggest that mating status triggers elimination of queens by workers, and that the colony origin and mating partner are unlikely to be important roles in elimination of queens.     

Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems,and their system specificity in mice

DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.     

Comparison of Stevia plants grown from seeds,cuttings and stem-tip cultures for growth and sweet diterpene glucosides

The growth and sweet diterpene glucosides of Stevia plants propagated by stem-tip cultures were compared with those of the control plants propagated by seeds. There was no significant difference between the two groups both in growth and in chemical composition. As for the contents of sweet diterpene glucosides, however, the clonal plants showed significantly smaller variations than the sexually propagated plants; they were almost as homogeneous as the plants propagated by cuttings. These results suggest that the clonal propagation by stem-tip culture is an effective method of obtaining a population of uniform plants for the production of sweet diterpene glucosides.