• Journal of Plant Biology
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• v.35 no.1
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• pp.77-84
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• 1992

In view of the well-established role of protein kinase C effector element in signal transduction of animal systems, the possibility of diacylglycerol (DAG) and its analog 12-O-tetradecanoylphorbol13-acetate (TPA) having an effect on auxin-induced growih of com coleoptiles was explored. Both DAG and TP A were found to promote cell elongation in the coleoptile tissue_ Treatment of tissue with these protein kinase C-activating agents resulted in increase in the growth rate over the control by about 300%. When 1M was applied to TPA-pretreated coleoptiles. auxin effect appeared synergistic. Morever. coleoptile growth was found to be inhibited by staurosporine and methylated TPA, both of which are known to specifically inhibit protein kinase C. Electrophoretic and autoradiographic patterns of soluble proteins from the coeoptiles indicated that either 1M or TPA tereatment resulted in increased phosphorylation of certain proteins of 205 Kd. 66 Kd and 32 Kd in size. The results obtained from the present work suggest that protein kinase C may be associated with auxin action on cell elongation in the corn coleoptile segments.gments.

• Animal cells and systems
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• v.1 no.4
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• pp.589-594
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• 1997

The neuronal voltage-sensitive calcium channels (VSCCs) are multisubunit complexes consisting of $\alpha_1,\;\alpha_2-\delta$ and $\beta$ subunits. Heterologous expression and biochemical studies have shown that the activity of VSCCs is regulated by their $\beta$ subunits in a $\beta$ subunit isoform-specific manner. To elucidate the $\beta$ subunit identity of the P/Q-type calcium channel encoded by an $\alpha_{1A}$ subunit, which is exclusively expressed in the Purkinje and granule cell of the cerebellum, we have examined the spatial and temporal expression patterns of $\beta$ subunits and compared them with those of $\alpha_{1A}$ subunit in the developing rat cerebellum. Reverse transcriptase- polymerase chain reaction (RT-PCR) and Northern blot analysis have shown that $\beta_4$ subunit mRNA was prominently expressed in the cerebellum and much more abundant than any other distinct $\beta$ subunits. RNase protection assay has further demonstrated that the expression of $\alpha_{1A}$ and $\beta_4$ subunits increased during cerebellar development, while the amount of $\beta_2$ and $\beta_3$ mRNAs did not significantly change. In addition, a $\beta_4$ transcript was present in cultured cerebellar granule cells, but not in astrocyte cells, and the level of $\beta_4$ mRNA expression increased gradually in vitro seen as in vivo. Based on the spatial and temporal expression patterns of $\beta_4$ subunit, we conclude that $\beta_4$ may predominantly associate, but probably not exclusively, with the $\alpha_{1A}$ subunit in rat cerebellar granule cells.

• Animal cells and systems
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• v.1 no.4
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• pp.641-646
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• 1997

Lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) has been known as an intercellular phospholipid messenger with a wide range of biological activities. In this study, the effect of LPA on both the proliferation and differentiation of rat E63 myoblasts has been investigated. In the serum-free Insulin-Transferrin-Selenium (ITS) media, the proliferation of E63 cells was largely restricted. Addition of LPA into the ITS media strongly promoted the cell proliferation and resulted in two to four fold increase of cell number. Furthermore, it appeared to increase the percent fusion in a dose-dependent manner up to 15 ug/ml. The synthesis of myosin heavy chain (MHC) was increased by LPA as well. These results indicate that LPA is able to promote both cell proliferation and differentiation in rat E63 myoblasts. Suramin, known to have uncoupling activity on growth factor-receptor interaction, was tested for antagonistic activity in myoblast proliferation and differentiation. Myoblasts grown in the ITS medium containing LPA were able to proliferate well even in the presence high concentration of suramin whereas myoblast differentiation was completely blocked by 30 ug/ml of suramin. The inhibitory effect of suramin on the myoblast differentiation was completely reversible by removing the suramin. This result indicates that the intracellular signaling pathway of LPA leading to cell proliferation might be distinct from that leading to cell differentiation on E63 myoblasts. Also, the antagonistic effect of suramin suggests that the differentiation activity elicited by LPA might be mediated by a specific G protein-coupled receptor.

• Animal cells and systems
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• v.4 no.3
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• pp.223-229
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• 2000

Limbs and supporting structures of an organism experience a full weight of its own when it lands from water, because neutral buoyancy in the aquatic habitat will be no longer available in the terrestrial world. Metamorphosis of anuran amphibians presents 8 good research model to examine how this transition from non-loading to weight-loading affects development of motor capacity at the time of their first emergence on land. Our video analysis of the transitional anurans, Rana catesbeiana, at Gosner stage 46 (the stage of complete transformation) demonstrated that the take-off speed increased 1.23-fold after the first six hours of weight-loading on the wet ground. It did not increase further during the following three days of loading, and was close to the level of mature frogs with different body mass. During development of larvae in deep water with no chance of landing through metamorphosis, both tension and power of a hindlimb anti-gravity muscle increased 5-fold between stages 37 and n. However, the muscle contractility increased more rapidly when the larvas could access the wet ground by their natural landing behavior after stages 41-42. Muscle power, one of major factors affecting locomotory speed, was 1.29-fold greater in the loaded than in the non-loaded larvae at the transitional stage. Thus, weight-loading had a potentially significant effect on the elevation of motor capacity, with a similar extent of increment in locomotory speed and muscle power during the last stages of metamorphosis. Such a motor adjustment of the froglets in a relatively short transitional period would be important for effective ecological interactions and survival in their inexperienced terrestrial life.

• Animal cells and systems
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• v.4 no.3
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• pp.287-292
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• 2000

The present work examined the role of gonadotropin-releasing hormone (GnRH) and dopaminergic drugs on the secretion of maturational gonadotropin (GTH II) in relation to testosterone m treatment. This study provides evidence that the plasma GTH II levels are increased by T treatment in precocious males, but not in the immature animal. In addition, GnRH analogue (GnRHa) alone significantly increased the plasma GTH II secretion in immature rainbow trout treated with T, as well as in T-treated and T-untreated precocious males. However, injection with either dopamine (DA) or domperidone (DOM; DA D2 receptor antagonist) alone did not alter the basal plasma GTH 11 secretion in all experimental groups. The secretion of GTH II in the T-treated precocious males was remarkably influenced by GnRHa or combination of dopaminergic drugs. Notably, the effects of dopaminergic drugs on GnRHa-induced GTH II secretion w8s prolonged by T in precocious males. In T-treated immature animals, GnRHa-induced GTH II secretion was Increased only by a dose DOM (10$\mu$g/g body n) but not by higher dose DOM (100$\mu$/g body wt). In the T-untreated immature rainbow trout, however, plasma GTH 11 secretion was not influenced by the same treatments. Therefore, these results indicate that DA may be acting indirectly by blocking the effect of GnRH on GTH II secretion in vivo. T may act to modulate the relative contribution by the stimulatory (GnRH) and inhibitory (DA) neuroendocrine factors, which would ultimately determine the pattern of GTH II secretion.

• Animal cells and systems
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• v.4 no.3
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• pp.299-304
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• 2000

In the present paper, the immunostimulatory effects of the extracellular products (ECP) from Mycobacterium spp. and various adjuvants on the non-specific immune responses of Nile tilapia, Oreochromis nilotica, were examined. Nile tilapia were immunized by injecting ECP of Mycobacterium spp. (strain TB40, TB267 or the type strain Mycobacterium marinum) into their swim bladders. A variety of adjuvants like as Freund s complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Titremax were similarly injected into additional groups of tilapia. The number of nitroblue tetrazolium (NBT)-positive cells observed in the swim bladder of the immunized fish was signigicantly increased by the fourth day post-immunization. By day 8, the numbers of NBT-positive cells were fewer in fish immunized with ECP from mycobacteria strains TB40 or TB267 than those immunized with ECP from M. marinum or fish injected with FCA or FIA. The level of Iysozyme activity detected in the serum of fish 40 alter immunization with ECP from various Mycobacterium spp. was also significantly higher than that found in the serum of the control fish. Head kidney macrophages showed enhanced reduction of NBT when cultured in vitro with 1 $\mu$ g/ml of ECP. Concentrations greater than this (10 or 100 $\mu$g/ml) were found to suppress the reduction of NBT by the macrophages. ECP from Mycobacterium spp. and the various adjuvants used in the study all appear to be good activators of the non-specific immune responses of Nile tilapia.

• Animal cells and systems
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• v.6 no.2
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• pp.145-151
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• 2002

Phylogenetic signal present in the mitochondrial 16S ribosomal RNA gene (16S rDNA) and the nuclear large subunit ribosomal RNA gene (28S rDNA) was explored to assess their utility in resolving family level relationships of the superfamily Tephritoidea. These two genes were chosen because they appear to evolve at different rates, and might contribute to resolve both shallow and deeper phylogenetic branches within a highly diversified group. For the 16S rDNA data set, the number of aligned sites was 1,258 bp, but 1,204 bp were used for analysis after excluding sites of ambiguous alignment. Among these 1,204 sites, 662 sites were variable and 450 sites were informative for parsimony analysis. For the 28S rDNA data set, the number of aligned sites was 1,102 bp, but 1,000 bp were used for analysis after excluding sites of ambiguous alignment. Among these 1000 sites, 235 sites were variable and 95 sites were informative for parsimony analysis. Our analyses suggest that: (1) while 16S rDNA is useful for resolving more recent phylogenetic divergences, 28S rDNA can be used to define much deeper phylogenetic branches; (2) the combined analysis of the 16S and 28S rDNAs enhances the overall resolution without losing phylogenetic signal from either single gene analysis; and (3) additional genes that evolve at intermediate rates between the 16S and 28S rDNAs are needed to further resolve relationships among the tephritoid families.

• Animal cells and systems
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• v.10 no.4
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• pp.227-231
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• 2006

Micro-deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so-called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZlike). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two-hybrid screening. A number of candidate Dazlinteracting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

• Animal cells and systems
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• v.10 no.4
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• pp.203-209
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• 2006

Azoles are widely used antifungal agents, which inhibit the biosynthesis of fungal cell-membrane ergosterol. In this study, using an amphibian follicle culture system, the effects of azoles on follicular steroidogenesis in frogs were examined. Itraconazole (ICZ), clotrimazole (CTZ) and ketoconazole (KCZ) suppressed pregnenolone ($P_5$) production by the follicles ($ED_{50};\;0.04_{\mu}M,\;0.33_{\mu} M,\;and\;0.91_{\mu}M$, respectively) in response to frog pituitary homogenates (FPH). However, fluconazole (FCZ), miconazole (MCZ) and econazole (ECZ) were not effective in the suppression of $P_5$ production. Not all the azoles examined suppressed the conversion of exogenous $P_5$ to progesterone ($P_4$) (by $3{\beta}$- HSD) or $P_4$ to $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) (by $17{\alpha}$-hydroxylase), or androstenedione (AD) to testosterone (T) (by $17{\beta}$-HSD). In contrast, CTZ, MCZ and ECZ in medium partially suppressed the conversion of $17{\alpha}$-OHP to AD (by C17-20 lyase) ($ED_{50};\;0.25{\mu} M,\;4.5{\mu}M,\;and\;0.7{mu}M$, respectively) and CTZ, KCZ, ECZ and MCZ strongly suppressed the conversion of exogenous T to estradiol ($E_2$) (by aromatase) ($ED_{50};\;0.02{\mu}M,\;8{\mu}M,\;0.07{\mu}M,\;0.8{\mu}M$, respectively). These results demonstrated that some azole agents strongly suppress amphibian follicular steroidogenesis and particularly, P450scc and aromatase are more sensitive to azoles than other steroidogenic enzymes.

• Animal cells and systems
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• v.10 no.4
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• pp.211-217
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• 2006

Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01-100 ${\mu}M$), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH-induced pregnenolone ($P_5$) production by the follicles ($EC_{50},\;4.0{\mu}M,\;25.6{\mu}M\;and\;5.7{\mu}M$, respectively ), and also suppressed the conversion of testosterone (T) to estradiol $17{beta}\;(E_2)\;(EC_{50},\;4.2{\mu}M,\;7.5{\mu}M\;and\;80.0{\mu}M) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as $C_{17-20}$ lyase and $17{\beta}$-hydroxysteroid dehydrogenase ($17{\beta}$-HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.