Promotion of Zygote Formation by Protein Kinase Inhibitors during the Sexual Development of Dictyostelium mucoroides

To analyze the possible involvement of protein kinases in the sexual development (macrocyst formation) of the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of several protein kinase inhibitors were examined. K252a, a potent inhibitor of protein kinase activities, promoted the sexual cell fusion, through enhancement of gamete formation. In contrast, staurosporine (structurally and functionally similar to K252a) inhibited markedly the progress of development including cell aggregation, thus resulting in the failure of cells to form mature macrocysts. The effective period of K252a was 5–7 hr after starvation, during which Dm7 cells could acquire fusion competence, and the inhibitory effect of cAMP on zygote formation was nullified by the co-application of K252a. Although KT5720 (a specific inhibitor of cAMP-dependent protein kinase) and W7 (a calmodulin inhibitor) had no effects on zygote formation when applied separately, their combined application enhanced zygote formation like K252a did. Neither calphostin C (a specific inhibitor of Ca²⁺-dependent protein kinase) nor herbimycin A (a specific inhibitor of tyrosine kinase) exerted a stimulative influence upon macrocyst formation. These results strongly suggest that the two signal transduction pathways mediated by cAMP-dependent protein kinase (PKA) and calmodulin are closely related to zygote formation, their blockage being favorable to zygote formation.     

Transient Gene Expression by SV40 Promoter Characterizes Sequential Differentiation of Embryohal Carcinoma F9 Cells into Primitive and Visceral Endoderm

Transient expression of the chloramphenicol acetyl-transferase (CAT) gene under the control of simian virus 40 (SV40), Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1α promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.     

A new snailfish of the genus Careproctus (Cottoidei: Liparidae) from the Pacific coast of southern Japan

Ichthyological Research - A new snailfish, Careproctus tomiyamai, is described on the basis of four specimens collected from Suruga Bay, Tosa Bay, and the Hyuga-nada Sea, southern Japan...     

基于Golden Gate技术的齐整小核菌转录单元高效组装系统

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术,以mobius assembly为基础,分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体,从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体,4个用于单转录单元组装的Level 1载体,4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力,为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。     

Intracellular distribution of the hydrophobic triterpene,bryonolic acid,in cultured cells of Luffa cylindrica L.

Summary Intracellular localization of bryonolic acid, an antiallergic pentacyclic triterpene, in cultured cells of Luffa cylindrica was investigated with reference to the sites of its biosynthesis and accumulation. The results of cell fractionation showed that bryonolic acid was mostly located in the cell wall fraction. The addition of FC-43 emulsion to the culture medium was found to cause the release of bryonolic acid from the cell wall into the medium without affecting cell growth and bryonolic acid production. Under this culture condition, ¹⁴C-labeled sodium acetate administered to the cells was rapidly incorporated into bryonolic acid which was then excreted into the medium within 10 min after administration. Electron microscopic observations suggested that spherical vesicles (ca 0.1 m in diameter) derived from the rough endoplasmic reticulum may be associated with the biosynthesis and excretion of this compound into the cell wall. Furthermore, the activity of 2,3-oxidosqualene cyclase, a key enzyme involved in the biosynthesis of bryonolic acid, was detected in the microsomal fraction containing the endoplasmic reticulum.Abbreviations BA bryonolic acid - ER endoplasmic reticulum - LS Linsmaier-Skoog - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PVPP polyvinyl polypyrrolidone     

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAP 6-isopentenylaminopurine - LS medium Linsmaier-Skoog medium - Growth medium LS medium containing 10^-6 M 2,4-D     

Recovery of aSasa tsuboiana population after mass flowering and death

The recovery process of aSasa tsuboiana population after a mass flowering and death in 1977 was investigated by 15 years of observation in the Hira Mountains, Kinki district, western Japan. Seed production was high (6600–13 800 seeds m⁻² inSasa plots and 3900 seeds m⁻² in a forest plot) but emergent seedling density was low (14–21 seedlings m⁻²), probably because of seed predation byMicrotus montebelli occurring between seed shedding and the next spring. The seedling density had decreased further by the next year and theS. tsuboiana population recovered from only a limited number of seedlings. In spite of such a low initial density, theS. tsuboiana population was able to regenerate successfully and attained the previous full stand height in 7–16 years.Miscantbus sinensis invaded and delayed the recovery ofS. tsuboiana in one plot, butS. tsuboiana became dominant as it caught up with the height ofM. sinensis. Seedling growth patterns, such as frequent tillering, the onset of rhizome extension in the early stage of seedling growth and frequent culm production from rhizomes, played important roles in the successful regeneration ofS. tsuboiana.     

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.