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[Adhesion and mobility of embryonic and tumoral cells].

Regulation of a number of adhesion molecules during neural crest cell migration was studied. The neural crest, a transient embryonic neural epithelium structure, undergoes mesenchymal transformation (epithelial-mesenchymal transition). The cells then migrate, giving rise to a variety of elements including the peripheral nervous system and melanocytes. During migration, neural crest cells do not express functional cell Adhesion Molecules but interact specifically with cell-binding domains in fibronectin molecules. A rat bladder carcinoma cell line was used as an in vitro model to study conversion of epithelial cells to a migratory fibroblast-like state. Conversion can be induced by culture on collagen or exposure to acidic Fibroblast Growth Factor (aFGF). Furthermore, constitutive fibroblast-like transformation can be induced by transfection with cDNA encoding aFGF. Growth factor-producing clones exhibit increased invasive and metastatic properties as compared with non-FGF-producing control cells. This model may provide increased understanding of the role of the different adhesion molecules in processes involving cell remodeling, such as tumor spread and development of metastases.

Cadherins↗

[Genetic monitoring in connection with the Chernobyl accident].

The complex investigation has been provided to control the genetically influenced processes after Chernobyl accident in some Ukrainian regions. Some dangerous trends in genetic populational status were revealed in the Ukrainian regions surrounding Chernobyl power plant allowing for the increasing number of congenital anomalies among the newborns and the spontaneous abortions as well as higher proportion in aberrations of chromosome type in human somatic and embryos cells, higher incidence of structural defects in embryonal bone system and higher, compared with control, proportion of children with intensive catabolism (R-protein homeostasis).

Abnormalities, Radiation-Induced↗

Retinal and lens damages observed in young rats undergoing protein malnutrition in selected stages of their growth.

Ocular damages of young rats undergoing protein malnutrition in selected stages of their growth have been valuated. Malnutrition was induced by a purified, hypoproteinic diet given to mothers or to newborns after their weaning. The histologic results pointed out that protein lack damages ocular structures different in embryonal origin: retina and lens. In fact, vacuolar degeneration of retinal optic fiber layer and cataract of lens were observed in experimental groups of rats. Only lens damage appeared completely reversible after a prolonged nutritional rehabilitation as for rats malnourished during fetal stage and lactation and then rehabilitated for 50 days. On the contrary retinal degeneration was still present in these animals even if some improvement could be observed.

Animals↗

A new method for early in utero experiments in rat embryos: endoscopy.

The merits of in vitro and in vivo techniques for experiments in rat embryos are discussed in this paper. Time limitation of culture, which is only feasible during 48 hours, up to day 13 post coitum (p.c.) is a major draw-back in the in vitro whole embryo culture. With the in utero operation technique used to date, no controlled experiments can be performed in rat embryos of 15 days p.c. and younger due to the high mortality of the embryos. Therefore a new technique has been developed, in which successful in utero operations can be performed as early as day 12 of gestation. Controlled micro-injection with the help of an endoscope can be given in any desired embryonic organ or structure. This paper describes this technique. Endoscopy in rat embryos of 12 days p.c. onwards has proven to be a new facility for in utero operations.

Animals↗

Suprapatellar plica synovialis: a common arthrographic finding.

The suprapatellar structures are often overlooked during double contrast knee arthrography. A plica synovialis suprapatellaris representing a remnant structure from the embryonic septum between knee joint and suprapatellar bursa was identified in 40 of 53 double contrast knee arthrograms. Symptoms including knee pain and clicking have been described in association with the presence of a plica synovialis. Only one of 40 patients in this series, however, was treated for the diagnosis of plica syndrome. Plica synovialis suprapatellaris is a common, usually incidental finding at knee arthrography and should not be mistaken for an adhesion or other capsular abnormality if the clinical findings of plica syndrome are absent.

Humans↗

Congenital abnormalities of the skin: future directions.

The inherited abnormalities of the skin are easily recognizable and readily accessible. They involve structures of different embryonic origins, are amenable to study in in vitro systems, are available for study in laboratory and domestic animal models, and are eminently suited to the type of interdisciplinary research presented at this workshop. Biochemists, geneticists, and clinicians in various disciplines all profit by this exchange of information. Ultimately, the gain is for the patient in the treatment and prevention of inherited dermatologic disorders.

Cell Differentiation↗

[Arterial vascularization of the lenticular nucleus].

The arterial vasculature of the lenticular nucleus was studied on 30 human brains the vascular system of which was injected with Indian ink mixed with gelose. The arteries of the lenticular nucleus belong to the group of anterior central arteries issued from the internal carotid artery and its terminal branches, except for the posterior cerebral artery. Examination of serial sections made translucent by the Spalteholtz method enabled us to follow the courses of these vessels within the lenticular nucleus. In this nucleus two vascular networks were present, with different vascular organization and density. These differences were due to the histological structure and specific embryonic origin of each part of the lenticular nucleus.

Adult↗

Inductive interactions during kidney development.

The conversion of intermediate mesoderm into renal tubular structures occurs throughout embryonic development from the formation of the nephric duct through to the generation of metanephric nephrons. Studies using an in vitro model system of renal epithelial morphogenesis indicate that the signals mediating tubulogenesis rescue intermediate mesoderm from programmed cell death, trigger spindle-shaped mesodermal cells to differentiate into epithelia, and lastly promote epithelial cell diversification and segmented tubule assembly. Recent data indicate that members of the Wnt family of developmental regulatory genes play an important role in the signaling pathway mediating renal epithelial morphogenesis.

Animals↗

Conservative management of an ovarian polyembryoma.

BACKGROUND: Polyembryomas are rare, immature germ cell malignancies characterized by numerous embryo-like bodies in association with mature and immature teratoma structures and primitive embryonic tissue. The purpose of this paper is to present a patient in whom surgical staging and postoperative serial tumor markers and imaging studies were used to determine management. CASE: A 43-year-old woman with a stage IA polyembryoma was followed with serial alpha-fetoprotein and hCG assays, as well as serial abdominal and pelvic computed tomography (CT) scans, following surgical staging and a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Chemotherapy was not given because the patient's tumor markers declined steadily into the normal range and imaging studies revealed no evidence of recurrent disease. CONCLUSION: Women with polyembryomas surgically staged and confined to one ovary may be followed with serial tumor markers and diagnostic imaging techniques to avoid aggressive cytotoxic chemotherapy.

Adult↗

Molecular medical approaches for alleviating infertility and understanding assisted reproductive technologies.

Fertilization is a precisely orchestrated cascade of events that results in the union of paternal and maternal genomes and in the establishment of mitotic potential of the zygote. To initiate embryonic development, the structures of the fertilizing sperm have to be disassembled and transformed into zygotic components by interactions with the cytoplasm of the egg. These interactions include the decondensation of the sperm nucleus into male pronucleus, the assembly of the zygotic centrosome, and the gathering of centrosomal proteins and sperm aster microtubules around the sperm centriole. Both the formation of the male pronucleus and the assembly of the zygotic centrosome are crucial steps required for pronuclear apposition and genomic union. The discovery of previously undetected fertilization failures that are due to defects in the assembly of the zygotic centrosome, abnormal pronuclear development, and compromised cytoskeletal dynamics enforces the development of new diagnostic strategies. Moreover, the introduction of new methods of infertility treatments, such as intracytoplasmic sperm injection and round spermatid nucleus injection into assisted human reproductive technology programs, emphasizes our lack of understanding of the cellular and molecular basis of human fertilization and evokes the need for additional experimentation. These efforts, however, are compromised by the sensitive nature of human embryo research and thus are severely restricted. Animal models that are reliable and cost-effective and that feature the characteristics of human fertilization have therefore been sought. Rodents such as the rat, mouse, and hamster are poor models owing to their maternal inheritance of the zygotic centrosome that is in strong contrast with the biparentally contributed assembly of the human zygotic centrosome during fertilization. Although rabbits are similar to humans from the standpoint of mitotic potential inheritance, information on postfertilization events in rabbits are lacking. Nonhuman primates represented by the rhesus monkey proved to be a reliable model for human in vitro fertilization and intracytoplasmic sperm injection, an advantage that is further emphasized by phyllogenetic similarity. In situations in which the high cost of primate research does not allow for large-scale experimentation (i.e., when large numbers of oocytes and embryos are needed), ruminants would be an ideal solution. Represented by the cow and sheep, domestic ruminants feature a fertilization strategy similar to that of the human. In addition, large numbers of gametes can be obtained wherever farms and slaughterhouses are accessible. Moreover, the detailed information on ruminant fertilization is strengthened by years of research and well-defined reproductive technology aimed at increasing the productivity of farm animals. Ruminants and rhesus monkeys have been extensively studied, and the data from these studies have been extrapolated in order to propose new strategies for the diagnosis and treatment of human infertility.

Animals↗

[Pulmonary blastoma in adults].

Pulmonary blastoma is a rare tumor of the lung consisting of immature mesenchyma and epithelial structures that can mimic the structure of the embryonic lung. In this article, we report a case of pulmonary blastoma occurred in an adult individual. We review the literature concerning this rare neoplasm that includes less than one hundred cases reported. We also describe the pathologic features of this disease which is microscopically divided in two classes: 1) lesions composed solely of malignant gland of embryonic appearance (WDFA: Well-Differentiated Fetal Adenocarcinomas), and 2) lesions with biphasic appearance, containing both neoplastic glands and either adult sarcomatous or embryonic mesenchyme. The adult pulmonary blastomas are considered as an entity distinct from childhood blastomas. This condition may be frequently asymptomatic; if symptomatic, it show the same clinical picture of a mass in the lung impinging on bronchi or pleura; the most usual symptoms include cough, hemoptysis, and chest pain. Chest radiography typically shows a peripheral or central lung opacity without preference for any lobe. The prognosis is poor, due to the high incidence of metastasis. Histologic class and clinical findings can be of prognostic value in pulmonary blastoma. We believe that an early diagnosis is essential: in our case, this allowed a complete resection of the tumor and the patient is still alive without recurrence 3 years after the surgical treatment.

Age Factors↗

Neuronal differentiation and synapse formation of PC12 and embryonic stem cells on interdigitated microelectrode arrays: contact structures for neuron-to-electrode signal transmission (NEST).

The development of neuron-microelectrode interfaces (neurochips) is highly desirable for the non-invasive recording of the cellular response to neuroactive drugs as well as the electrical stimulation of nervous tissue by implantable electrodes. A prerequisite for neuron-to-electrode signal transmission (NEST) is the formation of synapse-like contacts between the neuronal cell and the conductive surface of a microelectrode array. We attempted synapse formation by neuronal differentiation of rat pheochromocytoma cells (PC12) and blastocyst-derived murine embryonic stem cells (ES-J1) on interdigitated microelectrode arrays that were made of gold (Au), platinum (Pt), or indium tin oxide (ITO). PC12 or ES cells were in vitro differentiated by incubation with nerve growth factor (NGF) and forskolin, or by serum deprivation and treatment with basic fibroblast growth factor (FGF-2), respectively. On top of ITO electrodes, the neuronal cells extended extremely long processes that terminated in pili-like contact structures, which is typical for growth cone formation. ES cells differentiated into neurons as verified by immunofluorescence staining of MAP-2 and developed synapse-like junctions with the ITO electrode surface as indicated by synaptophysin staining. Differentiated PC12 and ES cells showed bona fide morphological characteristics of synaptic growth cones that were unprecedented in tissue culture. Cones formed by PC12 cells could be stimulated with KCI and carbachol as shown by uptake of FM1-43, a fluorescent marker for synaptic vesicle formation. In contrast to Electrical Cell Impedance Spectroscopy (ECIS) recordings, AC impedance spectrometry with differentiated PC12 cells settled on interdigitated microelectrode arrays revealed lower AC impedance than that with undifferentiated cells, indicating that the complex impedance is dependent on ion fluxes at the neuron-to-electrode contact surface.

Animals↗

[The mechanism of vasculogenesis: the critical role of transforming growth factor-beta 1 in the formation of vessel-like structures during the differentiation in vitro of murine embryonic stem cells].

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells. The above result is the first indication that the differentiation of tubular structures in embryoid bodies of ES-T cells may somehow be related to TGF-beta 1. To demonstrate further the role of TGF-beta 1 in the formation of vessel-like structures, the cultured ES-5 cells in the presence of added rhTGF-beta 1 were closely followed in the course of their differentiation. We have, thus, demonstrated the promoting effects of exogenous rhTGF-beta 1 in the formation of vessel-like structures, morphologically similar to those structures derived from ES-T6 cells, during the differentiation of ES-5 cells, both in monolayer culture, in three dimensional collagen gel and in embryoid bodies cultured on gelatin-coated tissue culture wells. Addition of suitable amount of anti-TGF-beta 1 monoclonal antibody IgG (TB21) to the culture medium of embryoid bodies of ES-T6 cells could effectively abolish the formation of vessel-like structures induced by retinoic acid. The percentage of the inhibition was very high, giving a figure comparable to that of atypical vessel-like structures formed in the control embryoid bodies from their parent ES-5 cells. The flat epithelial-like cells and round cells differentiated from embryoid bodies of ES-T6 cells were stained rather strongly for laminin and type IV collagen by immunofluorescent procedure. The above results indicate clearly that TGF-beta 1 is a crucial factor in organizing the differentiated derivatives (endothelial-like cells and their immediate progenitor cells) from ES-T6 cells to form vessel-like structures, and that the role of TGF-beta 1 in vasculogenesis might be performed, in part, through the modulation of the composition and organization of the extracellular matrix. In addition, the enhanced expression of bFGF mRNA in derivatives differentiated from both ES-5 cells treated with rhTGF-beta 1 and ES-T6 cells were detected by Northern blot analysis. Thus, aside from its effects on extracellular matrix, TGF-beta 1 might also modulate the bioactivity of bFGF in relation to the growth of vascular endothelial cells in the present system.

Animals↗

Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells.

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye-like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye-like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye.

Animals↗

Adaptation of sensory neurons to hyalectin and decorin proteoglycans.

Proteoglycans are abundantly expressed in the pathways of developing and regenerating neurons, yet the responses of neurons to specific proteoglycans are not well characterized. We have shown previously that one chondroitin sulfate proteoglycan (CSPG), aggrecan, is potently inhibitory to sensory axon extension in short-term assays and that over time, embryonic neurons adapt to aggrecan-mediated inhibition through the transcriptional upregulation of integrin expression (Condic et al., 1999). Here, we have compared the response of embryonic sensory neurons to structurally distinct CSPGs that belong to either the hyalectin (or lectican) family of large, aggregating proteoglycans or the decorin (or small leucine-rich proteoglycan) family of smaller proteoglycans. Both of these structurally diverse proteoglycan families are expressed in developing embryos and inhibit outgrowth of embryonic sensory neurons in short-term cultures. These results document a previously uncharacterized inhibitory function for the decorin-family proteoglycan biglycan. Interestingly, embryonic neurons adapt to these diverse proteoglycans over time. Adaptation is associated with upregulation of select integrin alpha subunits in a proteoglycan-specific manner. Overexpression of specific integrin alpha subunits improves neuronal regeneration on some but not all decorin-family CSPGs, suggesting that neurons adapt to inhibition mediated by closely related proteoglycans using distinct mechanisms. Our findings indicate that CSPGs with diverse core proteins and distinct numbers of chondroitin sulfate substitution sites mediate a similar response in sensory neurons, suggesting that increased integrin expression may be an effective means of promoting axonal regeneration in the presence of diverse inhibitory proteoglycan species in vivo.

Adaptation, Physiological↗

Cloning of a beta integrin subunit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata.

A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.

Amino Acid Sequence↗

Carbohydrate-mediated sorting in aggregating embryonal carcinoma cells.

The oligosaccharide structure carried by embryoglycan, Lex hapten, as well as E-cadherin, has been described to mediate Ca(2+)-dependent cell-cell adhesions in embryonal carcinoma cells. To examine the contribution of these two systems to intercellular adhesion, we analyzed aggregation properties of previously isolated embryoglycan-defective mutants of P19 embryonal carcinoma cells. Our data indicate that the absence of Lex and embryoglycan has no effect on homotypic cell aggregation. Pretreatment of the cells with E-cadherin-specific antibody reduced homotypic aggregation of both parental and mutant cells, suggesting that E-cadherin plays a major role in this system. When parental cells were mixed with mutant cells, the aggregates contained either parental or mutant cells; no heterotypic aggregation was observed. The absence of mixed aggregates formed between parental and Lex-or embryoglycan-negative mutant P19 cells suggests that carbohydrates are involved in cadherin-mediated cell sorting.

Animals↗

Umbilical cord agenesis in limb body wall defect.

The term "Limb Body Wall Defect" (LBWD) refers to a variable group of congenital defects having in common abdomino- or thoraco-schisis and limb deficiency. Three general pathogenic mechanisms have been proposed for this disorder: amnion rupture, vascular disruption, and embryonic malformation. We hypothesize that there are subsets of "Limb Body Wall Defect," which have similar structural abnormalities and a common pathogenesis. We report on five cases of LBWD that were selected by using more restrictive criteria. An infant or fetus was included if it had abdominoschisis with a broad attachment of skin to amnion at the site of the abdominal wall defect, limb defects, and umbilical cord agenesis. Autopsy detected additional common structural defects. All had evisceration of the gastrointestinal structures into the extra-embryonic coelomic space with structural abnormalities of the intestines. All had scoliosis, thoracic deformities, pulmonary hypoplasia, and structural abnormalities of the cloaca and urogenital ridge. Four of five had meningomyelocele. Three had exstrophy of the cloaca. The four females all had ovarian agenesis and incomplete Mullerian fusion. None had normal development of the external genitalia. We propose that the pathogenesis was a primary malformation of body wall closure, with abnormal fusion of the amnion, which had occurred in the first month of development.

Abdominal Muscles↗