Origin and development in vitro of shoot buds and somatic embryos from intact roots of Helianthus annuus x H. tuberosus

A variant clone of the tetraploid (2n = 4x = 68) interspecific hybrid Helianthus annuus x H. tuberosus derived by in vitro tissue culture showed a deviation from the usual pattern of organization of the plant body. This variant developed shoot-like structures and somatic embryos from intact adventitious roots of in vitro-grown plantlets. The morphogenetic structures were not normally able to differentiate complete plants. They did show cellular proliferation with the inception of additional secondary embryos, leaf-like structures and unorganized masses of callus. Nevertheless, some ectopic structures isolated from roots and transferred onto fresh basal medium without growth regulators were able to produce plantlets that exhibited the same phenotype as the original clone. Histological analyses demonstrate that they originate from cortical cells in association with the development of lateral root primordia.     

Expression of Dishevelled-1 in wound healing after acute myocardial infarction: possible involvement in myofibroblast proliferation and migration

One of our previous studies indicated that the expression of beta-catenin, which is the key factor of wnt-frizzled pathway, increased significantly in the ischemic area of the rat heart 7 days after myocardial infarction (MI). Together with the results of other recent studies, we made an assumption that wnt-frizzled pathway may be involved in the controlled cell proliferation and migration during repair processes after MI. To verify this assumption we tried to investigate the expression of another signal transduction molecule called Dishevelled in wnt-frizzled pathway during the wound healing process after MI. The left descending coronary arteries of rats were ligated to induce MI. Immunohistochemistry SABC method and in situ hybridization were performed to detect the expression of Dishevelled-1. The results showed, that one day after MI, Dishevelled-1 mRNA but not protein expression was detected in the cells at the border zone of the infarction area; 4 days after MI the expression of Dishevelled-1 increased exclusively and cytoplasmic Dishevelled-1 was observed not only at the border zone but also in the infarct area; 7 days after MI, it seems that the expression reached its peak, the positive staining even spread into the endothelial and smooth muscle cells of the newly formed and pre-existing blood vessels in the infarction area; after that the Dishevelled-1 expression decreased abruptly and could hardly be detected 28 days after MI. Thus cytoplasmic Dishevelled-1 may be involved in the controlled proliferation and migration of myofibroblasts and vascular endothelial cells, hence play a role during the wound healing process after MI.     

The MADS box gene AOM1 is expressed in reproductive meristems and flowers of the dioecious species Asparagus officinalis

MADS box genes are implicated in different steps of plant development. Some of them are expressed in vegetative organs. Most of them, however, are expressed in flower tissues and are involved in different phases of flower development. Here we describe the isolation and characterization of an Asparagus officinalis MADS box gene, AOM1. The deduced AOM1 protein shows the highest degree of similarity with FBP2 of Petunia hybrida and AGL9 (SEP3), AGL2 (SEP1) and AGL4 (SEP2) of Arabidopsis thaliana. In situ hybridization analyses, however, show that the expression profile of AOM1 is different from that of these genes: AOM1 is expressed not only in flower organs but also in inflorescence and flower meristems. These data indicate a possible function of AOM1 during flower development as well as in earlier stages of the flowering process. Asparagus officinalis is a dioecious species which bears male and female flowers on different individuals. AOM1, which is expressed very early during the process of flowering and has a similar expression profile in male and female flowers, does not seems to be involved in asparagus sex differentiation. Received: 3 July 2000 / Revision accepted: 4 August 2000     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Selective expression of a novel high-affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers

Within the flower, microsporogenesis represents a major sink for nitrogen, but knowledge on how the imported nitrogen is transferred from the anther cell layers to developing pollen is lacking. Here, we provide information on characterization of a transporter (AtLHT2) that might play an important role in partitioning of amino acids for microspore development. Biochemical analysis in yeast showed that AtLHT2 transports proline and aspartate with high affinity. However, other neutral and acidic amino acids act as strong competitors for proline and aspartate uptake indicating that AtLHT2 generally transports uncharged and negatively charged amino acids. Comparison of the apparent K _m values of AtLHT2 with previously characterized amino acid transporters clearly demonstrated that AtLHT2 represents a novel high-affinity system for neutral and acidic amino acids. Northern blot analysis showed strong expression of the amino acid transporter in flower buds. Cellular expression could be resolved by using RNA in situ hybridization and in situ RT-PCR methods, which localized AtLHT2 specifically to the tapetum tissue of the anthers. Developing pollen grains are symplasmically isolated from the sporophytic tissue and rely on the nutrients and other compounds secreted from the tapetum cells. Thus, the functional characterization of AtLHT2, together with our expression and localization studies, strongly suggest that in Arabidopsis flowers, AtLHT2 has a critical function in import of neutral and acidic amino acids into the tapetum cells for synthesis of compounds important for microspore structure and in transfer of organic nitrogen to the locule for pollen development.     

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.     

Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.     

The imprinted gene Delta like non-canonical Notch ligand 1 (Dlk1) is conserved in mammals,and serves a growth modulatory role during tissue development and regeneration through Notch dependent and independent mechanisms

Delta like non-canonical Notch ligand 1 (Dlk1) is an imprinted gene, mainly known for its involvement in adipogenesis, although it has been associated with many other stem cells/progenitors and is known to be widely expressed during organism development and tissue regeneration. In a systematic manner, we have outlined the overall expression pattern of Dlk1 in both man and mouse, and found Dlk1 to be expressed in tissues from all three germ layers. Yet, Dlk1 expression decreases along with increased differentiation as gestation proceeds and in most tissues Dlk1 is absent around birth. Thus, in adults, expression of Dlk1 is restricted to a few tissues and progenitor cells, but is re-expressed during disease and regeneration. Although diffferences exist, we found an overall conservation of Dlk1 expression between mouse and man, and conclude in that sense that the mouse is an appropiate model to study Dlk1. In agreement with the observed Dlk1 expression pattern, we found that the majority of published Dlk1 studies, report Dlk1 to have an inhibitory effect on both cell proliferation and differentiation, but the levels of the different DLK1 isoforms may be critical and have an impact on the overall outcome. This may also be an issue during tissue regeneration where several studies have reported Dlk1′s impact during skeletal muscle and liver regeneration without establishing the exact role. Likewise, the underlying mechanism of Dlk1 action is unknown, and seems to depend on both Notch dependent and independent pathways. However, from our data it is intriguing to speculate that the actual role of DLK1 may be to function as a checkpoint to slow down proliferation while forcing cells into the process of differentiation, and thus switch the cell/organ to a state of growth and hypertrophy. This may fit well with its reported impact on growth restiction and body size. Thus, our study which for the first time summarizes reported knowledge on Dlk1 in tissue development and regeneration as well as on the Dlk1 mechanism may provide novel insight to the general role of this remarkable imprinted gene in controlling cell growth, from which new hypotheses can be made in the field of stem cell biology and regenerative medicine.     

Expression of the RNA binding proteins,Mel-N1, Mel-N2, and Mel-N3 in adipose cells

Mel-N1 (murine embryonic lethal abnormal vision [ELAV]), a mammalian homolog of Drosophila ELAV, is an mRNA binding protein of the RNA Recognition Motif family. Studies with the human homolog, Hel-N1 have supported the hypothesis that Hel-N1, and its splice variant, Hel-N2 play a role in mRNA metabolism. Thus it becomes logical to extend this hypothesis to the murine variant Mel-N1 which has been described as a neuronal protein with a minor level of expression in the testis. Our current work expands the potential function for this protein through demonstration of expression of the full-length message and splice variants in adipose tissue as well as preadipocyte and adipocyte cell lines.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

番茄 DEAD-box RNA 解旋酶基因 SlDEAH1 的克隆及表达分析简

DEAD-box RNA解旋酶是一种特殊的RNA分子伴侣,参与了RNA代谢,包括前体RNA剪接、核糖体合成、RNA降解以及基因表达,并对植物的发育和抗性等也具有重要作用。根据已报道的拟南芥DEAD-box蛋白,通过同源比对,在NCBI据库中筛选得到一个DEAD-box RNA解旋酶同源蛋白,命名为SlDEAH1,并根据其基因序列设计特异引物,应用RT-PCR方法从野生型番茄（Solanum lycopersicum）AC⁺⁺中克隆得到了该基因的全长编码区序列。利用生物学网站、软件及实时荧光定量PCR方法,对其进行生物信息学、表达模式、胁迫及激素处理分析。结果表明：SlDEAH1包括2 073 bp的开放阅读框,编码690个氨基酸残基,其编码蛋白有9个保守结构基序,其所涉及到的ATP结合、ATP水解及RNA结合等功能对于解旋酶活性是至关重要的;表达模式分析表明SlDEAH1基因可能在野生型番茄萼片、叶片发育及果实成熟方面起到重要作用;高温、低温、脱水、伤害、盐胁迫不同程度的诱导了SlDEAH1的表达,但在根中该基因的表达受盐胁迫抑制;ABA、ACC、IAA、GA₃、MeJA和ZT均不同程度诱导了SlDEAH1的表达,其中ABA诱导效应最为明显。这些结果为进一步研究SlDEAH1在番茄发育和胁迫响应中的功能奠定了基础。     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-l/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-l/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-l/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice, mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced express     

Cloning and characterization of a mouse liver-specific gene mfrep-1, upregulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-1/LFIRE-1), a liver-specificprotein, is a member of fibrinogen superfamily that exerts various biological activities. However, the func-tion of HFREP-1/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mousefibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity toHFREP-1/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectivelyin mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA duringregeneration after 70% partial hepatectomy (PHx) in mice. mfrep-1 mRNA increased in the regeneratingliver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress theinduction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNAcontinued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression ofmfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistryassessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liverregeneration. These data suggested that MFREP-1 might play an important role in liver regeneration andbe involved in the regulation of cell growth.