DNA methylation variation in cloned mice

Summary: Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue‐specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue‐specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue‐specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned. genesis 30:45–50, 2001. © 2001 Wiley‐Liss, Inc.     

Migratory and proliferative effect of platelet-derived growth factor in rabbit retinal endothelial cells: Evidence of an autocrine pathway of platelet-derived growth factor

Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti-platelet-derived growth factor (PDGF) antibody, but not by an anti-bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF-BB and PDGF-AB dose dependently, but not by PDGF-AA, indicating that it was mediated by PDGF-β receptor-dependent pathways, and that the PDGF-like factor was PDGF-BB or -AB. In addition, PDGF-BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy. © 1994 Wiley-Liss, Inc.     

Effects of 2-bromo-α-ergocryptine on β-endorphin-induced growth hormone,prolactin and luteining hormone release in urethane anesthetized rats

Adult male rats were injected intraperitoneally either with saline or 2-Br- α-ergocryptine(CB-154)(10 ng/0.5 ml/rat) 30 min prior to an intraventricular injection of saline or β-endorphin (1 μg/10 μl or 5 μg/10 μl) and 30 min after β-endorphin, they were sacrificed by decapitation. Intraventricular injection of β-endorphin elicited significant increases in serum GH, prolactin and LH levels in a dose-related manner. Pretreatment with CB-154 inhibited the release of GH, prolactin and LH induced by β-endorphin. These results indicate that the stimulatory effects of β-endorphin on GH, prolactin and LH may be involved in an inhibition of dopaminergic mechanism in the central nervous system.     

A comprehensive software suite for the analysis of cDNAs

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initia- tion and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction. The software package is freely available under the GNU General Public License at http： / /www.g-language.org/ data/cdna/.     

Retardation of cell cycle progression of macrophages from G1 to S phase by ICAM-L from Leishmania

Leishmania, an obligate intracellular parasite of host macrophages, infects the macrophage through receptor-mediated phagocytosis that either activates or deactivates macrophages to eliminate the parasite or allow the parasite to grow intracellularly. ICAM-L, an intercellular adhesion molecule from L. amazonensis, results in lower MTT tests and proliferative responses of macrophages when incubated in vitro. The inhibition of cell proliferation, however, results from temporary retardation of the cell cycle progression at the G1 to S phase transition rather than cell death. The retardation is due to the upregulation of two CKI proteins, p21 and p27, in a p53-independent manner which, control the G₁ to S phase transition checkpoint.     

Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Differential display analysis reveals the expression of glutathione S-transferase ω and novel genes through an ITAM-containing receptor in ascidian immunocytes

The immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through antigen receptors in mammalian lymphocytes. We previously reported that an ITAM-containing receptor, ascidian hemocyte ITAM-containing receptor 1 (AhITAMR1), exists on the hemocyte surfaces of the ascidian Halocynthia roretzi, and is involved in both phagocytosis and hemocyte aggregation. In this study, we carried out differential display screening of upregulated genes during H. roretzi hemocyte aggregation and found that at least three genes are upregulated. One encodes glutathione S-transferase ω (GSTω), while the other two encode novel proteins. The expression of all three genes was induced by treatment with a specific monoclonal antibody against AhITAMR1, while their expression was inhibited by wortmannin, BAPTA-AM, and cyclosporin A. We also found that the expression of GSTω was induced by treatment with anti-T cell receptor antibody in mouse peripheral T cells. We propose that signal transduction pathways mediated by ITAM-containing receptors are conserved from ascidian hemocytes to mammalian T cells. The nucleotide sequence data reported in this study have been submitted to the DNA Data Bank of Japan (DDBJ) with accession numbers AB187220 for 18A-1, AB187221 for 20A, and AB187222 for 20G-1.     

Targeting signal 1 through CD45RB synergizes with CD40 ligand blockade and promotes long term engraftment and tolerance in stringent transplant models

The induction and maintenance of allograft tolerance is a daunting challenge. Although combined blockade of CD28 and CD40 ligand (CD40L)-costimulatory pathways prevents allograft rejection in some murine models, this strategy is unable to sustain engraftment in the most immunogenic allograft and strain combinations. By targeting T cell activation signals 1 and 2 with the novel combination of anti-CD45RB and anti-CD40L, we now demonstrate potent enhancement of engraftment in C57BL/6 recipients that are relatively resistant to costimulatory blockade. This combination significantly augments the induction of tolerance to islet allografts and dramatically prolongs primary skin allograft survival. Compared with either agent alone, anti-CD45RB plus anti-CD40L inhibits periislet infiltration by CD8 cells, B cells, and monocytes; inhibits Th1 cytokines; and increases Th2 cytokine expression within the graft. These data indicate that interference with activation signals one and two may provide synergy essential for prolonged engraftment in situations where costimulatory blockade is only partially effective.     

Binding effect of Cu(2+) as a trigger on the sol-to-gel and the coil-to-helix transition processes of polysaccharide, gellan gum

The binding effect of divalent cation Cu(2+) on the gelation process with a coil-helix transition in Cu(2+)/gellan aqueous solutions has been successfully elucidated by EPR, CD, and viscoelasticity measurements. Generally, Na-type gellan gum in aqueous solution can make gel when accompanied by an intrinsic coil-helix formation induced by hydrogen bonding between chains without any additional cations at T(ch)(-)(in) ( approximately 29 degrees C) with cooling temperature. An extrinsic coil-helix transition, induced by additional divalent cations in advance of the intrinsic sol-gel transition of gellan gum, is separately detected by CD measurement. The extrinsic coil-helix transition temperatures T(ch)(-)(ex) (>47 degrees C), which increased with the Cu(2+) concentration added, were nearly identical to the sol-gel transition temperature, T(sg), determined by the viscoelasticity measurement. Judging from the molar ellipticity by CD measurement and quantitative analysis of EPR spectra, it was elucidated that the helix forming process via divalent cations is composed of two steps ascribed to the different origins, i.e., a chemical binding effect via Cu(2+) ions in the initial stage and hydrogen bonds subsequently. Finally, we propose the coil-helix and the sol-gel transition mechanism initiated by the binding effect with the divalent cation, in which the partial chelate formation can cause local formation of helices and junction zones in the vicinity of the chelates at the initial stage of the process and stabilize the helices and the junction zones. On the other hand, the stabilized helices and junction zones can induce further formation and further stabilization of the Cu(2+)-gellan chelates. The mutual stabilization promotes the formation of three-dimensional network structure at the higher temperature than the intrinsic temperature for network formation.     

Intensity-dependent alteration of minnow (Pimephales promelas) behavior by a brain-encysting trematode

We examined the relationship between the numbers of brain-encysting trematodes (Ornithodiplostomum ptychocheilus) and the magnitude of altered behaviors in fathead minnows (Pimephales promelas). Because cysts develop within a brain region that integrates visual stimuli with motor response. we evaluated the standard optomotor response (OMR). Monitoring this task involved recording the time minnows spent following a spinning drum, on which alternating black and white stripes had been painted. Minnows were exposed to 0, 5, 20, 120, and 300 cercariae and then their OMR was evaluated at 2-wk postinfection. Surprisingly, only minnows that had high numbers of parasites (155 +/- 31 worms/fish) or low numbers of parasites (3 +/- 3 worms/ fish) differed significantly in their optomotor performance compared with controls. Reduced OMR of heavily infected minnows was positively correlated with reduction in minnow activity. In contrast, reduced OMR in lightly infected minnows was independent of host activity and was likely associated with the rapid development of parasite larvae within the optic tecta. The nonlinear relationship between parasite intensity and effect on host behavior was consistent with an earlier study, but the underlying mechanisms producing this pattern are unknown.