Effects of isotretinoin on the behavior of neural crest cells in vitro

Isotretinoin (13-cis-retinoic acid), an anti-acne medication, has been found to cause severe birth defects which affect the craniofacial elements, ear, heart, thymus, and central nervous system. Many of these structures receive contributions from the cranial neural crest. Here, we examine the possibility that these teratogenic effects are due to disturbances in neural crest development. Cranial and trunk neural crest explant cultures were exposed to different concentrations of isotretinoin and the cell morphology was monitored at daily intervals. Treated neural crest cells often became rounded or spindle shaped, separated from their neighbors, and frequently detached from the substrate or clumped together. In contrast, neural tube cells and cardiac fibroblasts were relatively unaffected by the drug. These results suggest that isotretinoin selectively affects neural crest cells by decreasing their cell-substratum adhesion.     

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

Genetic analysis of the microevolutionary processes in cattle populations (the example of the B locus of blood groups)

Dynamics of allele frequencies for the B-locus of blood groups in cattle populations and of a number of phenotype indexes in these animals was studied. It is determined that natural stabilizing selection and prezygotic selection, leading to a change in genetic structure, act in every population. The role of sires' influence on this process is insignificant.     

Current approaches to the synthesis and reconstruction of genetic material

Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated.     

Cytogenetic study of the effect of gossypol on mouse germ cells

The clastogenic and mutagenic activities of a new antifertility and antitumor agent gossypol were studied in the mouse male germ cells. Results of the present work indicate that at the doses 125 and 250 mg/kg the drug does not significantly increase frequencies of the micronuclei in the early spermatids and sperm head abnormalities. Hence, genotoxic influence can not be proposed as responsible for the antifertility effect of gossypol.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.     

The onset of the respiratory burst in human neutrophils. Real-time studies of H2O2 formation reveal a rapid agonist-induced transduction process

A real-time study of the initiation of the respiratory burst in human neutrophils was made. The cells were stimulated with fMet-Leu-Phe (fMLP) C5a, platelet-activating factor, leukotriene B4, phorbol myristate acetate (PMA), or ionomycin, and H2O2 production was determined by chemiluminescence. Identical average onset times (2.4 s) and closely comparable values for the apparent first-order rate constant (kapp) for the induction of NADPH-oxidase activity (0.21-0.29 s-1) were obtained following stimulation with fMLP, C5a, platelet-activating factor, or leukotriene B4, suggesting that different agonists act through a common transduction sequence. Much longer onset times and lower kapp values were obtained upon stimulation with PMA or ionomycin. Pretreatment with PMA consistently shortened the onset time of the neutrophil's responses to agonists by about 1 s. When H2O2 production was initiated with PMA, a subsequent stimulation with the agonist fMLP elicited an immediate response (onset time less than 0.2 s) which preceded further changes in fura-2-detected [Ca2+]i. The results are consistent with a mechanism in which agonist signals appear to be transduced by two sequences acting in concert--a rate-limiting one liberating Ca2+ and diacylglycerol and turning on the Ca2+/phospholipid-dependent enzyme protein kinase C, and an essentially instantaneous one which does not appear to require further changes in cytosolic Ca2+.