Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos

The concentration/effect relationship of bunazosin, a selective alpha-1-adrenoceptor antagonist of the quinazoline class, was established in hypertensive patients after a first dose of bunazosin and after 8 weeks of chronic dosing. The study was part of a placebo controlled, multicenter dose-finding trial with parallel group design. After a wash-out period and a 2-week placebo run-in phase, each 4 patients received either placebo, 3 mg, 6 mg or 12 mg bunazosin o.d. in a slow-release formulation. Concentration/effect analysis based on a sigmoidal Emax-model and the drop in the mean arterial blood pressure application of this model was feasible in 9/11 patients. The EC50 after the first dose was 4.6 +/_ 2.0 ng/ml and increased to 9.1 +/- 7.3 ng/ml at steady-state. The decrease in the average of the responsiveness parameter was mainly due to about three-fold, right-shift in the concentration/effect curves at steady-state in 3 patients, the remaining patients showed no or considerably lesser changes of the EC50. Whereas a significant correlation was observed between the pretreatment mean arterial blood pressure and the height of the maximal effect under bunazosin, no relationship appears between the bunazosin dose, the resulting plasma levels at trough and the corresponding effect.     

Effects of 13-cis-retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis)

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid-exposed Macaca fascicularis embryos. 13-cis-retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb-1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre- and post-otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.     

Expression of two even-skipped genes eve1 and evx2 during zebrafish fin morphogenesis and their regulation by retinoic acid

Growth and patterning during fin regeneration depend, like for fin development, on the integrated expression of homeogenes. In the present work we have studied, by in situ hybridization, the expression and regulation of two vertebrate homologs eve1 and evx2 of the Drosophila pair-rule even-skipped gene family. Upon amputation of pectoral and caudal fins, both genes, expressed transiently in the mesenchyme during early stages of fin development of these fins, are turned on. During the formation of the blastema they are transcribed first in the mesenchyme located underneath the wound epidermis and then, their expression is restricted to the regenerating rays regions. These expression patterns are developmentally regulated since both genes are no longer transcribed when the bony rays are differentiating. Exposure of the regenerates to retinoic acid (RA) modifies the boundaries of eve1 and evx2 expression: the signal is down-regulated in the ray region and up-regulated in the interray region. Moreover, expression is induced in the wound epidermis. These results indicate that eve1 and evx2 products are part of the molecular signals involved in pattern formation of the fin and fin rays in connection with outgrowth. RA might alter growth and morphogenesis of the regenerating fins by a fine regulation of these genes among others.     

Ectopic expression of SPARC in Xenopus embryos interferes with tissue morphogenesis: identification of a bioactive sequence in the C-terminal EF hand

A developmental chronometry hypothesis of early brain damage is suggested in which regions of the brain with a protracted course of postnatal development will be more vulnerable than earlier maturing areas to deleterious effects of early insult and, therefore, may become common sites of abnormality across many disorders originating in early childhood. Initial investigations of the cerebellum and frontal lobes are presented using MRI and neuropsychological measures. Planimetric measures of the cerebellar vermis (lobuli I-V and VI-VII) and pons, and neuropsychological frontal lobe measures were obtained from high functioning individuals with autism (A), survivors of acute lymphoblastic leukemia (ALL) with brain sequelae following radiation and chemotherapy, and from rigorously selected healthy controls (C). The neuropsychological results were clustered according to functions commonly related to frontal brain, posterior brain, and left and right hemispheres. The A and ALL groups, as compared to C, yielded modest but consistently reduced MRI measures for vermal lobuli I-V and VI-VII. Hypoplasia of lobuli VI-VII was more marked than I-V. Performance on neuropsychological tests for frontal lobe functions was generally depressed in both groups, with more severe deficits in A. Between-group differences in verbal, visual-spatial, and emotional-social skills are discussed. The cerebellar and frontal brain deficits that are present in both clinical groups (A and ALL) may be common to other developmental and acquired disorders of early childhood. Such joint manifestation of cerebellar and frontal lobe abnormalities is in agreement with the concept of cerebellar significance for the development of higher cognitive functions.     

Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype

Considerable evidence has demonstrated that retinoic acid influences the formation of the primary body axis in vertebrates and that this may occur through the regulation of Hox gene expression. In this study, we show that the phenotype induced by exogenous retinoic acid in the zebrafish can also be generated by the overexpression of Hoxa-1 following injection of synthetic RNA into the fertilised egg. The isolation, sequence and expression pattern of the zebrafish Hoxa-1 gene is described. We show that exogenously applied retinoic acid causes the ectopic accumulation of Hoxa-1 message during gastrulation in the hypoblast in the head region. Overexpression of Hoxa-1 following injection of RNA causes abnormal growth of the anterior hindbrain, duplication of Mauthner neurons in rhombomere (r) 2 and fate changes of r2 mesenchymal and neurogenic neural crest. These results are discussed in terms of the role of Hoxa-1 in controlling anterior hindbrain patterning and the relationship between expression of Hoxa-1 and retinoic acid.     

Correlation between loss of middle ear bones and altered goosecoid gene expression in the branchial region following retinoic acid treatment of mouse embryos in vivo

Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF-1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85alpha, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Gene expression and neuroblastoma cell differentiation in response to retinoic acid: differential effects of 9-cis and all-trans retinoic acid

Retinoic acid has considerable potential for the chemoprevention and chemotherapy of cancer. Neuroblastoma cells differentiate in response to retinoic acid in vitro, an observation that has led to clinical trials using either the 13-cis or all-trans isomers of retinoic acid. We review the effects of retinoic acid on neuroblastoma, and the potential involvement of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis retinoic acid is a ligand for RXRs, and we review recent data on the differential effects of 9-cis and all-trans retinoic acid on neuroblastoma differentiation and proliferation in vitro, and possible mechanisms of action via hetero- and homodimers of RARs and RXRs. Although there is uncertainty whether or not 9-cis retinoic acid produces its biological effects primarily via RXR homodimers, in vitro data suggest that this isomer of retinoic acid or stable analogues may have considerable potential for the treatment of resistant, disseminated neuroblastoma.     

Developmental expression of thrombin in zebrafish embryos: a novel model to study hemostasis

Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.     

Cloning of zebrafish neurofilament cDNAs for plasticin and gefiltin: increased mRNA expression in ganglion cells after optic nerve injury

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.     

Testosterone and progesterone level alterations in the adult rat after retinoid (retinol or retinoic acid) treatment (imprinting) in neonatal or adolescent age

Newborn rats were treated with a single dose of vitamin A (retinol), or with three doses of retinoic acid (in the 1st, 3rd and 5th days). Serum testosterone and progesterone level was measured in the four months old male and female rats, respectively. Retinol significantly decreased both hormone levels, however retinoic acid decreased the progesterone level only. In the second part of the experiments adolescent rats (in the 6th and 7th week after birth) were treated and measured similar to the newborns. In this case retinol significantly diminished testosterone level, without influencing the progesterone level. Retinoic acid decreased testosterone level and elevated progesterone level. The results demonstrate the long lasting effects of retinoid treatments at a neonatal or adolescent age, pointing also to the differences in the direction of the effects. Considering that previously the receptorial and sexual-behavioral effects of perinatal vitamin A treatments were observed, the experiments call attention to such harmful influences of perinatal vitamin A treatments, which are not manifested in morphological alterations.     

Response of four human ovarian carcinoma cell lines to all-trans retinoic acid: relationship with induction of differentiation and retinoic acid receptor expression

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR3 and OVCCR1 were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV1 cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RAR alpha was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RAR beta was not detected in any of the cell lines, while RAR gamma (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV1 cells.     

Abnormal regulation of retinoic acid receptor beta2 expression and compromised allograft rejection in transgenic mice expressing antisense sequences to retinoic acid receptor beta1 and beta3

Transgenic mice carrying antisense sequences common to the retinoic acid receptors (RAR) beta1 and beta3 were produced to examine roles of RARbeta1 and beta3 in the immune system. There were no significant changes of endogenous RARbeta1/beta3 expression at the mRNA level in T cells, B cells, and macrophages of the transgenic mice (AS-RARbeta1/beta3 mice). However, there was a decrease of RARbeta1/beta3 protein in the T cells, as expected. Interestingly, a drastic up-regulation (7-10 fold) of RARbeta2 messages and a significant decrease of RARbeta4 messages in AS-RARbeta1/beta3 macrophages were observed compared with that of nontransgenic macrophages. RARbeta2 protein in the transgenic macrophages was also up-regulated. This suggests that there is a dual feedback control for the RARbeta expression. A repressed RARbeta1 and beta3 expression, presumably at the protein level, likely leads to a compensatory up-regulation of RARbeta2 and repression of RARbeta4, which is less active than RARbeta2. The AS-RARbeta1/beta3 mice had no gross abnormality. However, they had compromised rejection response to cardiac allografts. The alloantigen-specific T cell cytotoxicity was reduced, and the macrophage population in the spleen was decreased in these mice. These defects likely contribute to the slower rejection response in the AS-RARbeta1/beta3 mice, the existence of other defects not being excluded. Our study has suggested that RARbeta1 and RARbeta3 are necessary to optimize immune responses.     

Effects of retinoic acid on lipoprotein lipase activity and mRNA level in vitro and in vivo

This study was designed to determine whether all-trans retinoic acid altered lipoprotein lipase (LPL) activity and mRNA levels in vitro and tissue LPL mRNA levels in vivo. Incubation of adipocytes or adipose tissue for up to 12 hr with 10(-6) or 10(-5) M all-trans retinoic acid did not decrease LPL activity. There was no change in LPL mRNA levels following 3 hr incubation of adipocytes with all-trans retinoic acid. Feeding all-trans retinoic acid for 4 days led to a significant decrease in adipose tissue LPL activity but no change in heart enzyme activity. Retinoic acid did not alter the increase in heart LPL activity observed with fasting. There were no changes in LPL mRNA levels in adipose tissue, heart or liver. Retinoic acid does not have an acute direct effect on adipose tissue LPL activity. The observed decrease in adipose tissue LPL activity in vivo is not due to alterations in mRNA levels and may be a secondary effect of retinoic acid.     

Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain

During development of the vertebrate hindbrain regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. We have taken a dominant negative approach to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, lacking kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells.     

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.