Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.     

Age of ovary determines remaining life expectancy in old ovariectomized mice

We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.     

Monitoring method for pre- and post-liver transplantation in patients with primary hyperoxaluria type I

Differential diagnosis of primary hyperoxaluria type I (PH1) may become difficult once end-stage renal failure and anuria have occurred. Here we describe a rapid and sensitive method to simultaneously quantify glycolate and oxalate in plasma using a stable isotope dilution and gas chromatography-mass spectrometry. The results are provided within 2 h. The linearity of the method was validated up to 200 micromol/l of these compounds and the inter-assay precision for glycolate and oxalate was 2.4 and 2.6%, respectively (n=5), when the control plasma was spiked with 50 micromol/l of glycolate and oxalate. For healthy subjects, 1.0 ml of plasma was required and 0.1 ml for the PH1 patients. Using this method, plasma levels in non-PH1 patients under hemodialysis and in healthy subjects were determined. This method proved to be useful when used for differential diagnosis of PH1 and for monitoring the plasma levels of glycolate and oxalate in two PH1 patients before and after dialysis and liver transplantation. Plasma glycolate in these patients was dramatically decreased after liver transplantation, but plasma oxalate decreased more slowly due to remobilization of the calcium oxalate stores deposited throughout the body.     

Coral reef rehabilitation through transplantation of staghorn corals: effects of artificial stabilization and mechanical damages

In order to develop and test a low-cost method of coral reef rehabilitation, the staghorn corals Acropora muricata and A. vaughani were transplanted to a shallow site with unstable substrate. To avoid abrasion, dislodgement and transport due to water movement, the transplanted corals were tied to string sections, which were connected at the seabed to form a grid. This created stability and improved the survival of the corals. The average increase in weight of live coral over 1 year was 56%, eight times more than the control treatment with unattached coral branches. This difference was mainly due to a reduced partial mortality among smaller coral fragments in the stabilized treatment. Survival was positively related to initial size among the loosely placed coral branches, whereas the attached treatment showed a negative relation between size and relative increase in weight of the surviving parts of the coral branches. Coral fragments were not significantly affected by severe physical damage simulating the effects of handling.     

Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells

Bovine embryonic stem-like cells (ES-like cells) appear to maintain a normal diploid karyotype indefinitely during culture in vitro and to express marker proteins that are characteristic of ES cells from mice, namely, alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1), STAT-3, and Oct 4. After proliferation of undifferentiated ES-like cells in vitro, some bovine ES-like cells differentiated to neural precursor cells, which were cultured in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). In addition, calves were successfully cloned using ES-like cells and the frequency of term pregnancies for blastocysts derived from ES-like cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells. Successful cloning from bovine ES-like cells should allow the introduction into cattle of specific genetic characteristics of biomedical and/or agricultural importance.     

Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells

Tumor-stroma interactions play a significant role in tumor development and progression. Alterations in the stromal microenvironment, including enhanced vasculature (angiogenesis), modified extracellular matrix composition, inflammatory cells, and dys-balanced protease activity, are essential regulatory factors of tumor growth and invasion. Differential modulation of stromal characteristics is induced by epithelial skin tumor cells depending on their transformation stage when grown as surface transplants in vivo. Tumor cells can regulate the development of a "tumor-stroma" via the aberrant expression of growth factors or induction of growth factor receptors in the stromal compartment. In this context, secretion of the hematopoietic growth factors G-CSF and GM-CSF, constituitively expressed in enhanced malignant tumors, may be good candidates for induction of a tumor stroma through their effect on inflammatory cells. Upon its induction, the tumor stroma will reciprocally influence the differentiation status of tumor cells resulting in a normalization of benign tumor epithelia and the maintenance of a malignant phenotype, respectively. In the HaCaT model for squamous cell carcinoma of the skin, stromal activation and angiogenesis are transient in pre-malignant transplants, however they remain persistent in malignant transplants where progressive angiogenesis is closely correlated with tumor invasion. While continued expression of VEGF and PDGF are associated with benign tumor phenotypes, activation of VEGFR-2 is a hallmark of malignant tumors and accompanies ongoing angiogenesis and tumor invasion. As a consequence the inhibition of ongoing angiogenesis by blocking VEGFR-2 signalling resulted in dramatically impaired malignant tumor expansion and invasion. Comparably, tumor vascularization and invasion was blocked by disturbing the balance of matrix protease activity caused by a lack of PAI-1 in the stromal cells of the knockout mouse hosts. A similar inhibition of tumor vascularization was caused by TSP-1 over-expression in skin carcinoma cells, which also blocked tumor invasion and expansion. On the other hand, when granulation tissue and angiogenesis were only transiently activated as a result of stable transfection of PDGF into non-tumorigenic HaCaT cells, the target cells formed benign, but not malignant, tumors. Collectively, these data show that tumor vascularization, providing intimate association of blood vessels with tumor cells, is a prerequisite for tumor invasion. A potential mechanism for this interrelationship may be the differential regulation of MMP-expression in tumors of different grades of malignancy. In vitro MMP expression did not discriminate between benign and malignant tumor cells unless they were co-cultured with stromal fibroblasts. However, in vivo regulation of MMP expression was clearly dependent on tumor phenotype. While MMP-1 and MMP-13 were down-regulated in benign transplants, they were persistently up-regulated in malignant ones. A tight balance between proteases and their inhibitors is crucial for both the formation and infiltration of blood vessels and for tumor cell invasion, thus again emphasizing the importance of the stromal compartment for the development and progression of carcinomas.     

Immunomodulation by the copolymer glatiramer acetate

Glatiramer acetate (GA; Copaxone, also known as Copolymer 1 or Cop-1), a copolymer of amino acids, is very effective in the suppression of experimental autoimmune encephalitis (EAE), the animal model for multiple sclerosis (MS), in various species including primates. The immunological cross-reaction between the myelin basic protein and GA serves as the basis for the suppressive activity of GA in EAE, by the induction of antigen-specific suppressor cells. The mode of action of GA is by initial strong promiscuous binding to major histocompatibility complex class II molecules and competition with MBP and other myelin proteins for such binding and presentation to T cells. Suppressor T cells induced by GA are of the Th2 type, migrate to the brain and lead to in situ bystander suppression. Clinical trials with GA, both phase II and phase III, were performed in relapsing-remitting MS (RRMS) patients, and demonstrated efficacy in reducing the relapse rate, decreasing MRI-assessed disease activity and burden and slowing progression of disability. GA is generally well tolerated and is not associated with influenza-like symptoms and formation of neutralizing antibodies seen with beta-interferons. It exerts its suppressive effect primarily by immunomodulation, and has recently shown ameliorating effect in a few additional autoimmune disorders as well as in graft rejection. At present GA is considered a valuable first-line treatment option for patients with RRMS.     

Mitochondrial dysfunction in liver disease and organ transplantation

The mitochondria play a crucial role in maintaining hepatocyte integrity and functions. Mitochondrial defects are either inherited or acquired. Mitochondria dysfunction occurs when the hepatocyte experience excessive physiologic stress. Its clinical presentation depends on the severity of the stress. It varies from mild abnormalities in liver biochemical tests to manifestations of acute or chronic liver failure. Mitochondria dysfunction is implicated in most liver disease and in early graft dysfunction after liver transplantation. This review will address the role of mitochondria in liver disease.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.