Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway.

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.

Retinoid signaling required for normal heart development regulates GATA-4 in a pathway distinct from cardiomyocyte differentiation.

Vitamin A is essential for normal embryonic cardiogenesis. The vitamin A-deficient phenotype in the avian embryo includes an abnormal heart tube closed at the sinus venosus and the absence of large vessels that normally connect the embryonic heart to the developing circulatory system. In vitamin A-deficient embryos the expression of cardiomyocyte differentiation genes, including atrial-specific myosin heavy chain, ventricular-specific myosin, and sarcomeric myosins as well as the putative cardiomyocyte specification gene Nkx-2.5, is not altered. However, the expression of transcription factor GATA-4 is severely decreased in the heart-forming regions of vitamin A-deficient stage 7-10 embryos. Significantly, GATA-4 transcripts are completely lacking in the lateral mesoderm posterior to the heart, in the area of the developing cardiac inflow tract that later displays prominent morphological defects, including a closed nonseptated heart lacking a sinus venosus. The administration of retinol to the vitamin A-deficient embryo restores GATA-4 expression and completely rescues the vitamin A-deficient phenotype. Our results indicate that GATA-4 is a component of the retinoid-mediated cardiogenic pathway unlinked to cardiomyocyte differentiation, but involved in the morphogenesis of the posterior heart tube and the development of the cardiac inflow tract.

Initial retinoid requirement for early avian development coincides with retinoid receptor coexpression in the precardiac fields and induction of normal cardiovascular development.

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. We demonstrate that all-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Retinoids provided to the vitamin A-deficient embryo up to the 5-somite stage of development, but not later, completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. Our studies provide strong evidence for a narrow retinoid-requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development.

[The evaluation of the cytotoxicity of preparations with anticarcinogenic action in human cell cultures]. / Otsenka tsitotoksichnosti preparatov s antikantserogennym deistviem v kul'turakh kletok cheloveka.

An approach for determination of cytotoxicity of chemical and biological drugs which combine the estimation of quality and quantity of cells after their treatment are proposed by the authors. The visual control of cellular minicultures permits one: 1) to register specific and pathologic alterations of cell morphology, 2) to trace their development in dynamics under the action of individual or complex drugs and 3) to choose the optimum time for the experiment fixation. The special staining of the treated cells and measurement of the optical density of absorbed histological dye permits one to make a conclusion about the changing of the cells quantity. A combined method of double estimation gives the opportunity to detect the artefacts taking place after staining the cells treated by some drugs and extracts of natural origin in high concentrations.

[The induction of mutations in 6-mercaptopurine resistance in Chinese hamster cells under the action of the recombinant plasmid pAins containing the human insulin gene]. / Induktsiia mutatsii rezistentnosti k 6-merkaptopurinu v kletkakh kitaiskogo khomiachka pod deistviem rekombinantnoi plazmidy pAins, soderzhashchei gen insulina cheloveka.

The mutagenic activity of the pUC19 bacterial plasmid DNA and the pAins recombinant plasmid DNA carrying human insulin gene has been investigated. Both pUC19 and pAins plasmid DNAs have been shown to induce the gene mutations in hprt locus of Chinese hamster cell line. The high level of the gene mutations (similar to the indices of the gene mutations induced by the chemical mutagens) has been in the focus of attention. The conclusion has been made concerning impossibility to use pAins plasmid DNA in the diabetes mellitus gene therapy. It is necessary to test the mutagenic properties of the DNA molecules produced for gene therapy of human inherited diseases.