Pax3-FKHR knock-in mice show developmental aberrations but do not develop tumors

Alveolar rhabdomyosarcoma is a pediatric disease specified by the recurrent chromosome translocations t(2;13) and t(1;13). These translocations result in the formation of the PAX3-FKHR and PAX7-FKHR fusion genes, which are thought to play a causal role in the genesis of this disease. Although PAX3-FKHR exhibits transforming activity in immortalized fibroblast cell lines, a direct role of this fusion protein in tumorigenesis in vivo has not been shown. We determined whether expression of Pax3-FKHR in the mouse germ line would render these animals prone to the development of rhabdomyosarcomas. By targeting FKHR cDNA sequences into the Pax3 locus of embryonic stem cells, we used these cells to generate mice carrying a Pax3-FKHR knock-in allele. Despite low expression of the knock-in allele, heterozygous offspring of Pax3-FKHR chimeric mice showed developmental abnormalities. These included intraventricular septum defects, tricuspid valve insufficiency, and diaphragm defects, which caused congestive heart failure leading to perinatal death. In addition, Pax3-FKHR heterozygous offspring displayed malformations of some but not all hypaxial muscles. However, neither newborn heterozygous pups nor their chimeric parents showed any signs of malignancy. We conclude that the Pax3-FKHR allele causes lethal developmental defects in knock-in mice but might be insufficient to cause muscle tumors.     

Turbidimetric method for quantitative determination of triton X-100 with silicotungstic acid

The Formation of Triton X-100-silicotungstic acid complex was studied. Quantitative turbidimetric determination of the detergent based on this process was suggested. This method allows to determining the complex formation at any wavelength in the range from 350 (epsilon 350 = 15,600 cm-1 M-1) to 600 nm (epsilon 600 = = 9090 cm-1 M-1). The calibration curve for Triton X-100 recorded at 350 nm is linear in the concentration range of 0 to 30 micrograms/ml. A sigmoid calibration curve was observed at longer wavelengths. A linear fragment of the calibration curve recorded at 600 nm was found at a concentration of Triton X-100 of about 5 micrograms/ml. The complex nature of calibration curves can be explained by heterogeneity of the complex dispersion.     

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.     

Immunologic activity of human lymph cells in the lymphocyte blast transformation reaction

The immunologic activity of lymph cells obtained by curative draining of the chest lymph drainage was studied in 14 patients with subhepatogenous jaundice of different origin. The lymphocyte blast transformation reaction showed that lymphocytes of the lymph can form blasts under the effect of phytohemagglutinin, antilymphocyte gamma-globulin and some bacterial antigens. The reaction of the highest intensity was recorded in lymphocyte cultures of the lymph collected for this purpose on the 2--3d day from the commencement of lymph drainage. Lymphorrhea produces a decrease in the capacity of stimulated lymph cells for blastformation. During lymph drainage no blastformation takes place in stimulated blood cell cultures of the patients. Therefore, the fundamental part of antigen sensitive lymphocytes is stored in the lymphoid tissue.     

The immunodiagnosis of thrombocyte membrane glycoprotein deficiencies: Glanzmann's thrombasthenia, Bernard-Soulier syndrome and GMP-140 protein deficiency]

Immunological methods were developed for the diagnosis of platelet membrane glycoprotein (GP) deficiencies. The number of membrane GP on platelet surface was determined as the binding of 125I-labeled monoclonal antibodies (mAB) directed against individual platelet GP. Total amount of GP in platelet lysate was assessed by immunoblotting with specific polyclonal antibodies. Methods were applied for patients with different thrombocytopathies. Binding of mAB VM16a, directed against GP IIb-IIIa was strongly decreased in patients with Glanzmann's thrombasthenia (0.5-14.5% of normal) and binding of anti-GP Ib mAB VM16d--in patient with Bernard-Soulier syndrome (0.5% of control) indicating the deficiencies of corresponding GP. In patient with gray platelet syndrome binding of both antibodies was not decreased but even increased. It was shown by immunoblotting that platelets from the patient with gray platelet syndrome contained normal amount of GP IIa, but strongly decreased amount of GMP-140 (14.5% of control)--membrane GP of platelet--granules.     

Elastolytic enzymes from Actinomyces fradiae 119]

Using ion-exchange chromatography and gel-filtration, elastase II, the main elastolytic component of protofradin (preparation of proteases of Act. fradiae 119), was purified to an electrophoretically homogeneous state. The enzyme is a serine proteinase with mol. weight of 17800 +/- 1000 and a pI greater than 10. The enzyme is stable at pH greater than 4,0 and exhibits its maximal activity towards elastin at pH 9,2. An analysis of elastolytic products revealed that the enzyme hydrolyses natural elactin of bovine nuchal ligament to form large fragments with mol. weights ranging from 25 000 to 80 000 and small oligopeptides. The elastolysis is ceased at the stage of formation of short-chained peptides, predominantly tripeptides. Elastase I is a minor component of protofradin and in its molecular weight and some enzymatic properties is similar to elastase II.     

ICI 182,780 has agonistic effects and synergizes with estradiol-17 beta in fish liver, but not in testis

Background  

ICI 182,780 (ICI) belongs to a new class of antiestrogens developed to be pure estrogen antagonists and, in addition to its therapeutic use, it has been used to knock-out estrogen and estrogen receptor (ER) actions in several mammalian species. In the present study, the effects and mechanism of action of ICI were investigated in the teleost fish, sea bream (Sparus auratus).     

Cloning of Mammals

A review of the development of the method of nuclear transplantation and cloning of animals with the use of nuclei of embryonic and adult cells is presented. We also present the results of studies of nuclear remodeling and reprogramming in the reconstructed oocyte and of cytoplasmic factors that control these processes.