Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Stress proteins by zinc ions in sea urchin embryos

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.     

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.     

The effect of 5-bromodeoxyuridine on the synthesis of nuclear and cytoplasmic DNA-binding proteins isolated from sea urchin embryos.

Newly synthesized DNA-binding proteins were isolated from the nuclei and, separately from, the cytoplasm of sea urchin mofula stage embryos. The presence of 5-bromodeoxyuridine during embryogenesis did not appear to alter the synthesis of either class of DNA-binding proteins. This result tends to argue that cell differentiation in early embryos is not regulated by differential synthesis of DNA-binding proteins. Sea urchin mofulae synthesize a broad range, by molecular weight, or cytoplasmic DNA-binding proteins which dissociate from sea urchin DNA-cellulose at relatively high salt concentrations (0.6-2.0 M NaCl). The most prominant of these apparently high-binding-affinity proteins has an approximate molecular weight of 33,000.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

Comparative distribution and embryotoxicity of hydroxyurea in pregnant rats and rhesus monkeys.

Hydroxyurea was given to pregnant rhesus monkeys and pregnant rats in regimens adjusted to produce similar degrees of teratogenicity, for the purpose of comparing the distribution of the drug in the females and their embryos. According, in rats 137 mg/kg/day ip on days 9-12 resulted in a drug half-life in maternal plasma of about 15 min and in embryos about 85 min, after the last injection; and in monkeys 100 mg/kg/days iv on days 23-32 resulted in drug half-life in maternal plasma estimated to be 120 min and in embryos 265 min, after the last injection. Using as a baseline of biological effects the minimal concentration known to inhibit DNA synthesis in rat embryos and cancer cells, namely 10(-4) M, it was calculated that the rat embryos in the present study were exposed to this level or more for approximately 12 h whereas the monkey embryos were exposed for approximately 100 h. Although the teratogenic effects were not identical in the two species, these data are interpreted to mean that rat embryos are teratogenically much more sensitive to hydroxyurea than monkey embryos. These observations have important implications in the selection of appropriate species for tests to estimate human teratogenic risks. The rat, which is currently the most widely used animal for such tests, displays sizeable differences from rhesue monkeys, which is one of the animals thought to be most like man in teratogenic susceptibility.     

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages.     

Salinity stress induced tissue-specific proteins in barley seedlings

Protein changes induced by salinity stress were investigated in two barley cultivars, California Mariout, a salt-tolerant variety and Prato, a salt-sensitive variety. Rapidly growing young barley seedlings were exposed to NaCl and the newly synthesized proteins were resolved on two dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. Salinity induces distinct protein changes in root and shoot tissues. In roots, the salinity effects are identical in both cultivars. First, salinity modulates the synthesis of two different sets of proteins, one of which is elevated, and the other, depressed. Second, six new proteins are induced all of which are low in molecular weight, 24 to 27 kilodaltons, with an isoelectric point range of 6.1 to 7.6. In contrast to roots, salinity induces cultivar-specific shoot proteins. Five new shoot proteins are induced whose molecular weights and isoelectric points fall within the range of 20 to 24 kilodaltons and 6.3 to 7.2, respectively. Three of the newly induced proteins are unique to Prato. In addition, salinity inhibits the synthesis of a majority of shoot proteins. The new proteins produced in roots and shoots are unique to each tissue and their induction is apparently regulated coordinately during salinity stress.     

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B₂ chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.     

Changes in liver nuclear sap proteins during chick embryo development

Summary Nuclear sap proteins from liver of 12-, 15-, 19-day-old embryos and 1-day-old chicks were resolved by one-and two-dimensional gel electrophoresis. Although the protein patterns from various stages of development have remarkable similarities, some qualitative and quantitative differences were found among these patterns. The most pronounced changes were detected in protein with molecular weight of 100 K which was very abundant in nuclei of 12-day-old embryos and disappeared in nuclei of older embryos and in protein with molecular weight of 40 K which rapidly diminished after hatching.     

Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.     

A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.     

Nuclear S1 proteins from the starfish Asterina pectinifera.

Nuclear S1 proteins are a group of proteins apparently ubiquitous in vertebrate cell nuclei. They were originally isolated at pH 4.9 from the supernatant of rat liver nuclei mildly digested with DNase I. In the present study, under the conditions identical to those employed for vertebrate cells, we identified two S1 proteins in the starfish Asterina Pectinifera. Their molecular weights are 47,200 and 39,000. This finding suggests widespread occurrence of S1 proteins in eukaryotes and their basic function in the cell nucleus.     

Embryotoxic effects of heterologous antisera against rat Reichert's membrane.

Parietal yolk-sacs of rat embryos at the fifteenth day of gestation were obtained by microdissection. A Reichert's membrane (RM) preparation was isolated by treating the parietal yolk-sacs with the chelating agent tetrasodium salt of ethylenediaminetetraacetic acid (EDTA) combined with mechanical shaking. Less than 1% of the membrane preparation was DNA and phosphorus contaminants. The membrane purity was also evaluated by electrom microscopic examination. Rabbit Ig G directed against the RM preparation when injected ip into ninth day pregnant rats produced malformations, fetal growth retardation and resorption. Fluorescent-labeled antibody localization studies demonstrated that the teratogenic antibodies localized in RM. It is postulated that RM antibodies induce teratogenesis by interfering with the function of RM.     

Phenytoin-induced stress protein synthesis in mouse embryonic tissue

Several proteins have been shown to be synthesized in response to various environmental stimuli, including treatment with teratogens. The role of these proteins in the teratogenic process is unknown. Pregnant A/J mice were treated with either a teratogenic or a non-teratogenic dose of the anticonvulsant drug, phenytoin (PHT). Protein synthesis in embryonic craniofacial (target) tissue or forelimb buds (non-target) was determined by incorporation of radiolabeled leucine and analysis by two-dimensional polyacrylamide gel electrophoresis. Synthesis of three proteins in target tissue and one protein in non-target tissue was stimulated by drug treatment. These results suggest that synthesis of specific stress proteins may serve as biomarkers of drug-target tissue interaction.     

视黄酸挽救的视黄酸合成酶Raldh2基因敲除鼠E10.75胚胎后肢发育正常

视黄酸合成酶Raldh2基因敲除鼠胚胎没有肢体的发育在胚胎E6.75-E 8.25期间,喂给怀孕母鼠含视黄酸（0.1 mg/g食物）食物后,Raldh2基因敲除鼠E10.75胚胎后肢形态正常,前肢发育较小.原位杂交结果表明,决定肢体近 远端轴发育的标志基因(marker gene)Fgf8,决定前-后轴发育的标志基因Shh以及后肢发育特异性基因Tbx4 和Pitx1在视黄酸挽救的Raldh2基因敲除鼠E10.75胚胎的后肢表达正常.上述结果提示,视黄酸可以挽救Raldh2基因敲除鼠E10.75胚胎后肢的正常发育.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.