Chromatin diminution, mitotic anomalies and cell death in early mouse embryos

The Feulgen-positive particles, similar to those in the nuclei, fragments of chromosomes and the whole chromosomes, and micronuclei have been found in the cytoplasm of early mouse embryos. Some morphological peculiarities of the interphase nuclei (extrusion of nucleoli-like structures from the nuclei, so called "partial" pycnoses) and mitoses (loss of chromosomes, the pattern of distribution of condensed chromatin similar to that with C-mitosis) are described. Dead cells were found in early blastocysts. The dynamics of these phenomena was traced in the course of development starting from the 4-cell stage to the blastocyst. The high frequency of enumerated findings enables the author propose the existence of the process of chromatin degradation in normally developing mammalian embryos, which causes genetic abnormalities in some cells of the embryo. This may constitute a step of differentiation for at least provisional structures of the embryo.     

Changes in elemental and isotopic composition accompanying larval growth and metamorphosis of the moor frog

A variety of early ontogenetic events of anuran species (growth, structural and biochemical diversification, metamorphosis) offers a unique opportunity to evaluate the effectiveness and application limits of mass spectrometry method for the analysis of metabolic and transformation events in developing organisms. The dynamics of relative carbon and nitrogen contents and stable isotopes of these elements during larval development in the period of metamorphosis climax and after its conclusion in moor frog specimens developing in their natural habitat and in vitro on a referent diet are traced. A decrease in C/N ratio and enrichment of the tissues with heavy stable isotopes of carbon and nitrogen during embryonal and larval development (prior to the beginning of independent feeding) indicates the increase in the portion and variety of proteins, accompanied by consumption of yolk lipids. The relative nitrogen content increase and C/N ratio decreases with the growth and development of independently feeding tadpoles, which indicates surpassing increase of the portion of proteins in tissues. In growing tadpoles, the rates of tissue renewal in general and rates of protein metabolism in particular affect the kinetics of changes of tissue isotope composition, which approaches isotope composition of the consumed food. A decrease in С/N ratio in the bodies of metamorphs during mass tissue decomposition is indicative of continuing reconstruction of larval organs and growth of anlage of definitive organs. Significant increase of C/N ratio and depletion of liver samples by heavy carbon isotopes are associated with intensive synthesis and reservation of lipids within the organ. Strong enrichment of metamorphs’ tissues with heavy nitrogen isotope indicates the substitution of ammoniotelic type of nitrogen metabolism by urotelic type. Decrease in C/N ratio and enrichment of tissues by heavy carbon isotope may be connected to intensive oxidation of lipids, which supports the growing energy costs of terrestrial underyearlings. Relative contents of heavy nitrogen isotope in the tissues of underyearlings does not change compared to the tissues of metamorphs.     

Akt mediates sequestration of the beta(2)-adrenergic receptor in response to insulin.

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Herein we examined the signaling downstream of insulin receptor activation, focusing upon the role of 1-phosphatidylinositol 3-kinase and the serine-threonine protein kinase Akt (also known as protein kinase B) in the internalization of beta(2)-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the beta(2)-adrenergic receptor, implicating Akt in downstream signaling to the beta(2)-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor by Akt in vitro identified Ser(345) and Ser(346) within a consensus motif for Akt phosphorylation. Double mutation (i.e. S345A/S346A) within this motif abolishes insulin counterregulation of beta-adrenergic stimulation of cyclic AMP accumulation as well as insulin-stimulated sequestration. Furthermore, expression of constitutively activated Akt (T308D/S473D) mimics insulin action on cyclic AMP responses and beta(2)-adrenergic receptor internalization. Expression of the dominant-negative version of Akt (K179A/T308A/S473A), in contrast, abolishes both insulin counterregulation of the cyclic AMP response as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in beta-agonist-induced desensitization.     

Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.     

Age changes in rat vasomotor reflexes and sympathetic neuron ultrastructure following chemical sympathectomy]

Prolonged administration of guanethidine (20 mg/kg) to newborn rats caused a marked reduction in the number of cells in stellate ganglia. The administration of guanethidine for 14 days decreased the amount of cells to 30% of the normal (partial sympathectomy), and for 28 days--to 0.5% (complete sympathectomy). At the age of two months the blood pressure pressor reflexes to asphyxia and femoral nerve stimulation were absent in both groups of the sympathectomized animals. These responses, however, were restored in the partially sympathectomized animals at the age of four months. No restoration took place in the completely sympathectomized animals. The electron microscopic studies of neurons in the partially sympathectomized animals showed the presence of a great number of neurofibrils. According to literature data this fact was typical of cells in which an active growth of axon fibers took place.     

Determination of Anesthetics Used in Adult Ophthalmology by the Multisensory Stripping Voltammetry Method in Tear Fluids

Biophysics - For effective dosage of ophthalmic drugs, it is necessary to assess the dynamics of changes in their concentration in the tear fluid over time. This paper provides examples of...     

Affinity and catalytic heterogeneity and metal-dependence of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with systemic lupus erythematosus

It was shown using enzyme-linked immunosorbent assay (ELISA) that titers of antibodies against human myelin basic protein (hMBP) in systemic lupus erythematosus (SLE) patients 4.2-fold higher than in healthy individuals, but 2.1-fold lower than in patients with multiple sclerosis (MS). Approximately 86% electrophoretically and immunologically homogeneous SLE immunoglobulin Gs (IgGs) purified using several affinity resins including Sepharose with immobilized hMBP specifically hydrolyze only hMBP but not many other tested proteins. Several rigid criteria were applied to show that the hMBP-hydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors. In contrast to MS IgGs, abzymes from SLE patients are more sensitive to ethylenediaminetetraacetic acid and less sensitive to specific inhibitors of serine-like proteases. We present the first evidence demonstrating a significant diversity of different fractions of SLE IgGs in their affinity for hMBP-Sepharose, the ability of IgGs to hydrolyze hMBP at different optimal pHs (5-10) and be activated by different metal ions: Ca(2+) > Mg(2+) ≥ Co(2+) ≥ Fe(2+) ≥ Ni(2+) ≥ Zn(2+) ≥ Cu(2+) ≥ Mn(2+) . Combinations of Ca(2+) + Mg(2+) and Ca(2+) + Co(2) lead to a significant increase in the antibody proteolytic activity as compared with Ca(2+) , Co(2+) , or Mg(2+) ions taken separately. Our findings suggest that the immune systems of individual SLE similar to MS patients can generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons and play an important role in pathogenesis not only MS but also SLE patients.