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The aim of this study was to investigate the effects of Rad9 mutants with impaired DNA mismatch repair (MMR) function on the tumorigenesis of colorectal cancer.The colorectal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 (hRad9) gene in these samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The plasmid of pFLAG-hRad9 (L101M) was constructed following the QuickChange mutagenesis procedure and transfected into mRad9-deleted mouse cells (mRad9(-/-) cells). The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function.Mutation from Leu to Met at the residue 101 (L101M) of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9(-/-)+ L101M cells (mRad9-deleted mouse cells with ectopic expression of L101M hRad9 gene) was (34.0±5.6)%, which was significantly lower than that in the mRad9(-/-)+ hRad9 cells [mRad9-deleted mouse cells with ectopic expression of hRad9 gene, (48.0±7.5)%, P0.05]. After N-nitroso-N-methylurea (MNU) treatment, the survival rate of mRad9(-/-)+ L101M cells was (33.7±5.9)%, which was significantly higher than that in the mRad9(-/-)+ hRad9 cells [(21.3±4.7)%, P0.05]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of hRad9 gene with mutation at the target residues of post-translational modification in mRad9(-/-) cells also led to a reduced MMR activity.Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.

In this study, bioinformatics analysis, tissue distribution and developmental expression pattern of lipoprotein lipase (lpl) and hepatic lipase (lipc) in zebrafish Danio rerio are reported. In adult D. rerio, lpl was highly expressed in liver. This is remarkably different from the tissue expression pattern of LPL in mammals, which is not detected in the adult liver. The expression of lipc was liver specific, which is consistent with that in mammals. During embryogenesis, lpl mRNA was increased gradually in concentration from 0.5 hpf (hour post fertilization) to 6 dpf (days post fertilization), but lipc was not expressed at the early stage of the embryo until 3 dpf. In situ hybridization further displayed the expression pattern of lpl mainly restricted to the head region including cells surrounding the mouth opening, branchial arches, pectoral fin and lateral line neuromast, whereas lipc was mainly restricted to the liver and part of head regions including lens. This lays a foundation for further investigation of lpl or lipc function and evolution in fishes.

Prolactin regulatory element binding (PREB) protein belongs to the family of WD-repeat proteins which are regulatory and versatile proteins for diverse functions. In this study we have shown the expression pattern of the planarian Dugesia japonica PREB-related gene (DjPreb) during embryonic development by whole-mount in situ hybridization. Genomic analysis reveals that the DjPreb gene consists of two exons and one intron. Expression of the DjPreb mRNA was not observed as early as in stage 3 embryos. DjPreb positive signal was first found in stage 4. It is expressed in some embryonic cells in the periphery of the embryo. The number of DjPreb positive embryonic cells grows in stage 5. DjPreb is expressed in the dorsolateral regions and part of the anterior regions in stage 6. In stage 7, DjPreb positive signals are detected in the dorsolateral regions along the A-P axis and from stage 8 to the juvenile stage DjPreb mRNA is strongly expressed not only in the differentiating tissues of the anterior and posterior regions, but also in the parenchyma of the dorsolateral regions, and generates the gradient in the head of the juvenile. These results on the DjPreb expression pattern suggest its potential role in the specification of many cell types; in particular, DjPreb may play an essential role in spatial and temporal regulation during the head and tail formation and the anterior/posterior patterning formation.

In this study we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5'-UTR and a 60 bp 3'-UTR. Phylogenetic analysis shows that DjPreb belongs to PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient of expression with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.

Establishment of a cell-mediated gene transfer system has potential as a new breeding technique for commercially valuable fishes. As an important step toward developing an inter-species chimera, cells from blastula-stage embryos of rosy barb (Puntius conchonius) were transplanted into zebrafish (Danio rerio) blastula-stage embryos to observe the development of the recipient. From the total of 473 transplants obtained only a fraction of 13 chimeras appeared perfectly normal after one month. Over two in normal 13 chimeras showed some characters from the donor cells with scarce pigmentation. This is the first successful inter-species study on zebrafish by using blastula cell transplants from rosy barb both belonging to the same family cyprinidae.

The relationship between microglial activation and dysmyelination/demyelination was analyzed in a long-lived myelin mutant, the Long Evans shaker (les) rat, which exhibits early dysmyelination and a later loss of abnormal myelin sheaths. A microglial reaction characterized by progressive morphological transformation and increasing cell density was localized exclusively to white matter during postnatal 2-4 weeks, suggesting a microglial response to dysmyelination and oligodendroglial pathology. A further microglial reaction as marked by microglial expression of MHC II and a concomitant expression in the brain and spinal cord of mRNA for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) began around 4 weeks when the remaining myelin was lost. Ultrastructurally, activated microglia ingested numerous myelin figures, suggestive of active phagocytosis. Thus, this study indicates that microglial reaction is graded in chronic neurological disorders and suggests that MHC II expression marks a functional change of activated microglia.

To understand how the differentiation of stem cells to oligodendroglial progenitors is regulated, we established cultures of neural stem cells from neonatal rat striatum in the presence of epidermal growth factor (EGF) as free-floating neurospheres that were then exposed to an increasing amount of B104 cell-conditioned medium (B104CM). The resultant cells proliferated in response to B104CM but no longer to EGF. In vitro analysis and transplantation studies indicated that these cells were committed to the oligodendroglial lineage, and they were thus referred to as oligospheres. Further characterization of their expression of early markers, cell cycle, migration, and self-renewal suggests that they were pre-O2A progenitors. RT-PCR analysis indicated that the oligosphere cells expressed mRNAs of platelet-derived growth factor alpha receptor in addition to fibroblast growth factor receptor but not EGF receptor; the latter two receptor mRNAs were expressed by neurosphere cells. Thus, the progression of stem cells to oligodendroglial progenitors is likely induced by factors in B104CM.

Transplantation repair of demyelinating lesions is restricted because relatively few cells can be introduced at only a limited number of sites. Repair could be enhanced by stimulating division of transplanted cells and by directing migration to multiple or distant lesions. This article demonstrates that transplanted oligodendroglial progenitors proliferate more when co-grafted with growth factor-secreting cells, yet retain the capacity to form myelin. Transplanted glial cells also migrate preferentially toward the growth factor-secreting cells when the two are implanted at separate sites. This opens avenues to examine growth factor actions on glia in vivo and improves the prospects for human remyelination therapies.

Transplantation of myelin-forming cells is a promising strategy for the treatment of myelin disorders. In this study, transplantation of glial cell progenitors into the cerebral ventricles of the embryonic myelin-deficient rat, a model of Pelizaeus-Merzbacher disease, was performed to assess the ability of these cells to incorporate into the developing brain and produce myelin. The donor cells migrated into the white and gray matter and produced myelin at widespread sites ranging from the corpus callosum and optic nerve to the cerebellum. These data suggest that myelin repair might be achieved by intraventricular delivery and transependymal incorporation of myelin-producing cells. Because these cells were genetically transduced to express a reporter gene, similar ex vivo manipulation with genes known to promote survival, migration, or proliferation of the transplanted cells could be used to enhance repair. Such a therapeutic strategy may be feasible in patients with inherited myelin disorders or in multiple sclerosis, particularly where the lesions are periventricular.

We have previously shown that oligodendroglial progenitors (OP) can be generated from multipotent rat neural precursor cells. We now report the generation of a homogeneous culture of canine OP from neural precursor cells. In non-adherent cultures, homogeneous OP cultures were obtained in 6-8 weeks of treatment with B104 cell conditioned medium (B104CM). In adherent cultures where astrocytes grew as a layer of substrate, colonies of OP invariably appeared at 10-14 days in vitro (DIV) and the colonies were expanded as free-floating spheres (oligospheres), in the presence of B104CM, suggesting that astrocytes facilitate the generation of canine OP. The oligosphere cells were characterized by self-renewal in the presence of B104CM and by terminal differentiation into oligodendrocytes after withdrawal of B104CM. Transplantation studies indicated that the extensively expanded oligosphere cells retained myelination capacity. The oligospheres thus provide a valuable source for experimental cell therapy studies.

We reported previously that stem cell factor (SCF) is produced mainly by neurons and that its receptor (c-kitR), encoded by the protooncogene c-kit, is expressed in microglia, suggesting that SCF/c-kitR signaling may be involved in neuron-microglia interactions. We now report that SCF supports microglial survival in cultures, maintains them in process-bearing morphology, and inhibits microglial proliferation induced by colony stimulating factor-1. SCF potentiates microglial expression of the mRNAs of nerve growth factor, brain-derived neurotrophic factor and ciliary neurotrophic factor, and downregulates microglial expression of the inflammation-associated cytokines, tumor necrosis factor-a (TNF-alpha), and interleukin-1beta (IL-1beta). SCF potentiates lipopolysaccharide-stimulated, but attenuates interferon-gamma TFNalpha mediated expression of the mRNAs of IL-1beta and TNF-alpha. The SCF-induced expression of neurotrophin mRNAs is enhanced by the addition of lipopolysaccharide (LPS) but is reduced by IFNgamma. The interactions between SCF and LPS or IFNgamma in the regulation of inflammation-associated cytokine gene expression are accompanied by the differential regulation of c-kitR in microglia. These observations suggest that SCF/c-kitR signaling modulates microglial activity.

Tob is a member of the PC3/ BTG1/Tob family of vertebrate tumor suppressor genes; its expression is known to inhibit proliferation of cells in vitro, but its possible roles during normal development have not been investigated previously. The present study concerns the structure and developmental expression of AmphiTob in an invertebrate chordate, amphioxus. This is the first investigation of any Tob gene during embryological development. The 311 amino acid AmphiTob protein is similar to vertebrate Tob but lacks the C-terminal PQ-rich domain of the latter. In early embryos of amphioxus, in situ hybridization first reveals AmphiTob expression in the hypoblast at the gastrula stage on the likely dorsal side of the embryo. During subsequent development, expression is seen in several tissues of the ectoderm, mesoderm, and endoderm. The most striking expression domains are in the developing somitic musculature and dorsal nerve cord. In the medial wall of each somite, AmphiTob is expressed strongly by cells destined to differentiate into the axial trunk muscles; this pattern persists until late in the larval stage, evidently because undifferentiated cells are continually becoming myogenic as the muscles grow. Nerve cord cells conspicuously transcribe AmphiTob from the late neurula until the early larval stage: Expression occurs in a few cells scattered along the nerve cord and in a group of cells located in the cerebral vesicle (in a region presumably homologous to the vertebrate diencephalic forebrain). During development, an intense and transitory transcription of AmphiTob may be an early event in cells exiting the cell cycle in preparation for differentiation.

We have characterized the cellular localization of stem cell factor (SCF) and c-kit receptor (c-kitR) in the adult mouse nervous system in situ and in culture by using immunocytochemistry. We found that SCF is largely confined to the neuronal population in normal brain, whereas c-kitR is expressed by glial cells as well as some neurons. We also found that astroglia at an early stage of culture (7 days in vitro) are strongly SCF positive and weakly c-kitR positive. Microglia in cultures express both SCF and c-kitR, but the immunostaining of SCF is weak and diffuse when microglia are cultured in the presence of colony stimulating factor-1. Northern blot analysis confirmed the expression of mRNAs of c-kit and SCF in cultured neurons, astroglia, and microglia. The addition of recombinant SCF to astroglia in culture upregulates the expression of mRNAs of nerve growth factor, brain derived neurotrophic factor, and ciliary neurotrophic factor. These observations suggest that SCF/c-kitR signaling is involved in neuron-neuron as well as neuron-glia interactions.

The results of the experiment showed that Daphne genkwa was slightly different from its processed product in the content and color of essential oil. The result of stimulation experiment showed that the essential oil had certain stimulation to eye-conjunctive of rabbits. The stimulation diminishes after processing with vinegar.