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PubMed1983

Unusual structure of the chicken embryonic alpha-globin gene, pi'.

We report the DNA sequence of the globin locus encoding the chicken embryonic alpha-globin, pi'. The structure differs significantly from that of the two chicken adult alpha-globin genes, alpha A and alpha D, as well as from that of previously studied adult alpha-globin genes in that the introns of the pi' gene are substantially larger than those in adult alpha-globin loci. In contrast, the pi' introns are structurally similar to the only other expressed embryonic alpha-globin gene reported to date, the human zeta gene. While completing the sequence of the pi' gene, we determined that only one chromosomal locus within the chicken genome hybridizes to a pi' central exon probe. These data lead to the conclusion that if the equimolar chicken embryonic alpha-globin polypeptides, called pi and pi', are indeed independently transcribed, then that transcription occurs from alleles of the same gene; however, we favor the possibility that the pi gene does not actually exist. This conclusion is drawn from the observation that the two chromosomal alleles of embryonic alpha-globins (represented by recombinant bacteriophage lambda CaG5 and lambda CaG7) both encode pi'.

Alleles↗
PubMed1998

Crystal structure of a human embryonic haemoglobin: the carbonmonoxy form of gower II (alpha2 epsilon2) haemoglobin at 2.9 A resolution.

The production of recombinant embryonic haemoglobins via a yeast expression system has enabled structural and functional studies to be conducted on these proteins. As part of a programme aimed at understanding the properties of the embryonic haemoglobins we have crystallized the human alpha2 epsilon2 (Gower II) embryonic haemoglobin in its carbonmonoxy form, and determined its structure by X-ray crystallography. The structure was solved by molecular replacement and refined at 2.9 A to give a final model with R-factor=0.185 and Rfree=0.235. The Gower II hemoglobin tetramer is intermediate between the adult R and R2 states, though closer to R2. The tertiary structure of the conserved alpha subunit is essentially identical when compared to that found in the adult (alpha2 beta2) and fetal (alpha2 gamma2) hemoglobins. The embryonic epsilon subunit has a structure very similar to that of the homologous adult beta and fetal gamma subunits, although with small differences at the N terminus and in the A helix. Amino acid substitutions can be identified that may play a role in the altered response of the Gower II haemoglobin to allosteric effectors, in particular chloride ions. The reduced chloride effect is thought to be the primary cause of the higher affinity of this embryonic hemoglobin in comparison to the adult molecule.

Heme↗
PubMed2006

Effects of hesperetin on vessel structure formation in mouse embryonic stem (mES) cells.

OBJECTIVE: The present study investigated the effects of hesperetin on vessel structure formation in mouse embryonic stem (mES) cells with regard to whether hesperetin acts as an antioxidant or pro-oxidant. Some flavonoids enhance antioxidant systems while increasing oxidative stress in the body. METHODS: After their differentiation into endothelial-like cells for 10 d, mES cells were treated with 1 to 100 muM of hesperetin for 24 h. RESULTS: Hesperetin efficiently inhibited the formation of vessel-like tubular structures consisting of platelet-endothelial cell adhesion molecule-1-immunoreactive cells and significantly (P < 0.05) increased the generation of reactive oxygen species in a concentration-dependent manner. Although glutathione (in its reduced and oxidized forms) in mES cells was not affected by hesperetin, the 8-iso-prostaglandin F2(alpha) content was decreased. In addition, cytotoxicity-induced hesperetin was not found; lactate dehydrogenase release and cell viability were determined as an index of cell damage. CONCLUSION: Taken together, the present study shows that hesperetin inhibits vessel formation by pro-oxidant means and suggests its potential as an antiangiogenic agent.

Animals↗
PubMed1980

[Experimental models of embryonic nephroblastomas (Wilms' tumor)].

Histological structure and histochemical characteristics of renal tumours induced in rats either by N-nitrosodimethylamine (DMNA) or N-nitrosoethylurea (NEU) are described. The rats treated with oral administration of DMNA developed 1 nephroblastoma and 176 mesenchymal tumours similar to mesoblastic nephroma. In rats receiving the transplacental treatment with NEU 24 mesenchymal tumours and 5 nephroblastomas corresponding to the epithelial variant of human Wilms' tumour occurred. Histologic and histochemical findings show that the mesenchymal neoplasms contain the epithelial component in the form of preexisting renal tubules. These tubules are similar to those of the normal kidney and strongly differ from the primitive tubular structures of embryonic rat kidneys and nephroblastomas.

Animals↗
PubMed2002

A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression.

A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure. Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC). Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product. This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody. Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c. Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form). Collision-induced dissociation (CID)-MS(2) showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)(3)-Gal-Glc-Cer and (NeuAc)(4)-Gal-GalNAc, respectively. These structures were confirmed by CID-MS(3) of the corresponding peaks. Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galbeta1-3GalNAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. This ganglioside was designated as GS1c. A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration. The present study suggests that GS1c may play a specific role in early development of chicken brain.

Animals↗
PubMed1998

Alterations of myocardial contraction associated with a structural heart defect in embryonic chicks.

Ablation of cardiac neural crest at stages 8-10 produces a structural heart defect (persistent truncus arteriosus, PTA) in embryonic chicks. PTA is associated with decreased myocardial contractility, as indicated by decreased left ventricular ejection fraction. We compared the force of small ventricular strips from normal and defective chick hearts. In intact muscle, ablation of the neural crest leads to a 30-50% decrease in twitch force at any level of extracellular Ca2+ (0.45-20 mM) at embryonic days (ED) 7 and 15, relative to sham-operated controls. These differences could reflect defects at the level of the contractile apparatus and/or in the excitation-contraction coupling process. To distinguish changes of the contractile apparatus, we used detergent skinned preparations. The maximal Ca(2+)-activated force (Fmax) at ED15 was not significantly different between control and experimental embryos. At ED 7, however, Fmax was reduced by 36% in experimental preparations. Electron-micrographs showed that the organization and orientation of the myofibrils was similar in experimental and control ventricles. At ED 14, however, the average myofibrillar diameter was significantly increased in experimental ventricles. The content of the major myofibrillar proteins (myosin heavy chain, actin, and tropomyosin), determined from polyacrylamide gel electrophoresis and Coomassie Blue staining, normalized to total protein, was not statistically different in experimental and control ventricles at ED7. At ED15, however, content of these proteins was doubled in experimental ventricles. These data suggest a possible defect of the contractile apparatus at both ED 7 and 15, since the ratio of Fmax/myosin is reduced in the experimental hearts.

Animals↗
PubMed1998

Structural basis of an embryonically lethal single Ala --> Thr mutation in the vnd/NK-2 homeodomain.

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to -5 degreesC in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25 degreesC. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

Amino Acid Sequence↗
PubMed1983

The embryonic development of ear-tufts and associated structural head and neck abnormalities of the Araucana fowl.

Developing embryonic structural abnormalities of ear-tufted embryos of the Araucana fowl are described. These abnormal structures are peduncle, cleft, ear opening, tympanic membrane, and columella auris. The structural abnormalities are believed to be due to the early incomplete fusion of the hyoid and mandibular arches from the distal part of the ear opening to the neck area.

Animals↗
PubMed2005

[Comparative morphometric analysis of rat embryonic and fetal pulmonary structures after general hypothermia].

The applied model of morphological assessment of the lung at strictly dated stages of embryonic (gestational day 14) and fetal (gestational day 20) development permitted to specify major planimetric parameters of organ parenchyma, the magnitudes of form-factors of the objects studied. On the basis of morphometric criteria, two main phenotypical variants of rat lung development were established. The factor of hypothermia, by modifying the limits of normal development, "typifies" its variants already in embryonic and perinatal periods with the participation of preacinar regions. This phenomenon is a manifestation of an individual intrauterine preadaptation by the formation of individual variants of "effect of readiness" to the challenge by a similar factor after birth with the object of probable minimal expenditures for the organism.

Animals↗
PubMed1985

Association of maternal and newly synthesized ribosomes with membranous noncytoskeletal structures in Xenopus laevis embryonic cells.

In Xenopus laevis embryos a high concentration of both KCl and 0.5% DOC (sodium deoxycholate) is needed for maximal extraction of ribosomes and polysomes. We studied the nature of the structures that keep ribosomes and polysomes immobilized within the cytoplasm of embryonic cells at cleavage through tailbud stages, using various combinations of a low-salt buffer (20 mM KCl), a high-salt buffer (500 mM KCl), 0.5% DOC, and 0.5% Triton X-100. With a low-salt buffer and 0.5% DOC, but not Triton X-100, 80S ribosomal monomers and polysomes were liberated from the cytoplasmic rapidly sedimenting structures (RSS) to the soluble fraction. With a high-salt buffer (500 mM KCl), ribosomes were solubilized as 60S and 40S subunits together with about one-half of the total polysomes. When cells were homogenized in a low-salt buffer with added inhibitors of the cytoskeleton (cytochalasin B or colchicine), the majority of polysomes but not ribosomes were solubilized. These results provide evidence for the following conclusions. 1) Polysomes are bound to cytoskeletal structures in Xenopus embryos, but ribosomes, both maternal and newly synthesized, are associated with membranous noncytoskeletal structures. 2) The membranous structures consist of two compartments, one high-salt sensitive and the other high-salt resistant. 3) Ribosomes of the high-salt resistant group increase in amount with developmental stage and appear to be the precursor to the ribosomes of the high-salt sensitive group.

Animals↗
PubMed2000

The prepro vasoactive intestinal contractor (VIC)/endothelin-2 gene (EDN2): structure, evolution, production, and embryonic expression.

Murine vasoactive intestinal contractor (VIC) and its human analog endothelin-2 (ET2) are potent vasoactive hormones composed of 21 amino acids. To study the structural characteristics of the VIC/ET2 gene (HGMW-approved symbol EDN2), we isolated the full length of the mouse VIC gene. Sequence analysis indicates that a biologically active mature VIC peptide is produced from a 175-residue precursor protein; preproVIC (PPVIC). Several remarkable similarities of the PPVIC gene to the human preproendothelin-1 gene strongly suggest that the two genes have arisen from a common progenitor by gene duplication. Transfection of ACHN adenocarcinoma cells with the cDNA resulted in the production of VIC peptide. VIC production was increased by the deletion of the 3'-untranslated region, which contains an AU-rich mRNA destabilizing sequence. Increased PPVIC gene expression during the late embryonic stage suggests an important function in development. This study provides the basis for disruption and regulation analysis of the gene, which may lead to a better understanding of VIC/ET2's physiological significance.

Amino Acid Sequence↗
PubMed2006

Formation of gut-like structures in vitro from mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.

Animals↗
PubMedDate not supplied

Use of embryonic stem cell-derived endothelial cells as a cell source to generate vessel structures in vitro.

Embryonic stem (ES) cells could potentially serve as an excellent cell source for various applications in regenerative medicine and tissue engineering. Our laboratory is particularly interested in generating a reproducible endothelial cell source for the development of prevascularized materials for tissue/organ reconstruction. After developing methods to isolate highly purified (>96%) proliferating populations of endothelial cells from mouse embryonic stem cells, we tested their ability to form three-dimensional (3-D) vascular structures in vitro. The ES cell-derived endothelial cells were embedded in 3-D collagen gel constructs with rat tail collagen type I (2 mg/mL) at a concentration of 10(6) cells/mL of gel. The gels were observed daily with a phase-contrast microscope to analyze the time course for endothelial cell assembly. The first vessels were observed between days 3 and 5 after gel construct formation. The number and complexity of structures steadily increased, reaching a maximum before beginning to regress. By 2 weeks, all vessel-like structures had regressed back to single cells. Histology and fluorescent images of the vessel-like structures verified that tube structures were multicellular and could develop patent lumens. We have shown that endothelial cells derived, purified and expanded in vitro from ES cells sustain an important endothelial cell function, the ability to undergo vasculogenesis in collagen gels, indicating that endothelial products derived in vitro from stem cells could be useful in regenerative medicine applications.

Animals↗
PubMed1996

Chicken Pax-1 gene: structure and expression during embryonic somite development.

Recent mouse genetic studies have implicated Pax-1, a paired-box-containing gene, in sclerotomal differentiation and vertebral body formation. To investigate Pax-1 function in somitic sclerotomal differentiation in the chick embryo, we have cloned the chicken Pax-1 gene, and its full length cDNA, and characterized its temporal and spatial expression pattern during somite development. Sequence analysis shows that chicken Pax-1 is highly homologous to murine and human Pax-1 genes with respect to the putative DNA-binding paired-box domain and the octapeptide domain. Northern analysis using probes derived from the paired-box domain and a unique non-paired box sequence of chicken Pax-1 detected 2-kb mRNA transcript. The expression profiles of Pax-1 were examined by in situ hybridization and Northern analysis. The first detectable expression of Pax-1 is seen in the most caudal epithelial somite. As the somite matures, Pax-1 expression takes on a medial distribution, thus corresponding to but preceding the emergence of the sclerotome. In the more mature, rostral somites (stage V and older), Pax-1 expression is found to be progressively localized first to the ventral-medial regions, and then to the caudal-ventral-medial quadrant of the mature somite. This pattern strongly supports the notion that Pax-1 expression is involved in somitogenesis and sclerotomal differentiation, and that it is subsequently a characteristic of the caudal half of the sclerotome, the presumptive precursor of vertebral cartilage. Northern analysis substantiated this expression profile and further revealed that the level of somitic Pax-1 expression increases as a function of embryonic development. Finally, we subjected chicken embryos to controlled heat shock treatment to perturb somite formation and segmentation. The pattern of Pax-1 expression in the anomalous somitic structures generated by controlled heat shock further supports a functional role for Pax-1 in somite development.

Amino Acid Sequence↗
PubMed1988

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells.

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.

Animals↗
PubMed1980

Projections to the superior colliculus from the dorsal hypothalamic area and other prosencephalic structures derived from the embryonic subthalamic longitudinal band of the diencephalon.

After injections of an aqueous solution of horseradish peroxidase (HRP) restricted to the superior colliculus of the cat, labeled neurons were found ipsilaterally in the dorsal hypothalamic area, lateral hypothalamus, zone incerta, reticular thalamic nucleus and ventral lateral geniculate. In most cases labeled neurons were observed in the same structures of the contralateral side. Some scattered neurons were observed in other hypothalamic structures and the globus pallidus. Most of these prosencephalic formations are subthalamic derivatives, thus having a common embryologic origin.

Animals↗
PubMed1998

Comparison of the liver and biliary duct development in man and in the rat at the end of the embryonic period.

Hepatic and biliary structures present during embryonic Carnegie stage 23 were analyzed and compared in OFA-IOPS rat and human embryos. The group of embryos was composed of 3 rat specimens-crown rump length (CRL) 16 mm, age 16 days 5 hours post coltus- and of 1 human embryo- CRL 31 mm, age 57 days PC-. The specimens were submitted to serial histological sections with graphic reconstruction. In both species, the basic architecture of liver parenchyma was established including parenchymal cords or plates alternating with hepatic sinusoids. Veinous channels were clearly defined. The intrahepatic biliary system, referred as ductal plates (DP) developed into the periportal connective tissue while the portal spaces were not yet existing. DP was recognized either as a single layered cuboidal structure or as a double layered one dilated to form mature tubular ducts. The latter layout was more frequent in the rat. Except for gallbladder which is never present in the rat, similarity and presence of the same hepatic structure in both species at the end of the embryonic period (stage 23) suggest that the rat is a good experimental model for liver development and will be useful to understand the mechanism of congenital anomalies and specially the involvement of ductal plates in the congenital diseases of intrahepatic bile ducts.

Animals↗