Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down's syndrome.

Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down's syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.

Developmental and pathological expression of peroxisomal enzymes: their relationship of D-bifunctional protein deficiency and Zellweger syndrome.

We present the developmental changes of peroxisomal enzymes, catalase, L-bifunctional protein (L-BF) and D-bifunctional protein (D-BF), in the normal brains, and patients with D-BF deficiency, a new peroxisomal disease. D-BF immunoreactivity was observed in controls as early as 13 gestational weeks (GW) and increased with maturation. The adult pattern with fine granule staining of somata and dendrites became apparent in adolescence. L-BF appeared at 20 GW in the cerebral cortex and Purkinje cells and positive glia appeared early in the white matter at 17 GW, and then increased with age. Catalase-positive neurons were identified in the same manner as L-BF, D-BF deficiency in both fetus and infant showed markedly diminished enzyme immunoreactivity. Patients demonstrate reduced D-BF expression. Zellweger syndrome shows decreased expression for the three proteins. This study shows that the peroxisomal enzymes may be closely related to neuronal maturation and gliogenesis in human brain and to disturbance of neuronal migration as seen in Zellweger syndrome significant. D-BF deficiency may exhibit a range of symptoms during the neonatal and early infantile periods some of which may be similar to Zellweger syndrome.

Epignathus: report of a case with successful outcome.

Epignathus is an extremely rare form of teratoma that arises from the palate or pharynx in the region of the basisphenoid (Rathke's pouch). This condition is associated with a high mortality rate caused by severe airway obstruction in the neonatal period, thus requiring prenatal planning and prompt surgical treatment after birth. The authors describe a case of a giant epignathus that was successfully resected followed by an uneventful recovery.

Aberrant methylation of an imprinted gene U2af1-rs1(SP2) caused by its own transgene.

Genomic imprinting refers to the parental allele-specific expression of genes. The precise mechanism underlying this phenomenon, which may involve DNA methylation, is not yet known. U2af1-rs1(SP2) is an imprinted gene expressed from the paternal allele and is methylated on the maternal allele. Here we report an artificial system in which expression and methylation of the endogenous imprinted gene U2af1-rs1 can be affected by interaction with its own transgene in the testis. We suggest that there is a mechanism in male gametogenesis by which the U2af1-rs1 gene is kept unmethylated to be expressed in the offspring in addition to a mechanism in female gametogenesis by which the U2af1-rs1 gene is methylated and is not expressed in the offspring.

Developmental analysis of cardiovascular system of 45,X fetuses with cystic hygroma.

There have been few pathological investigations of 45,X embryos and fetuses from a developmental point of view. Since most 45,X embryos and fetuses are lost prenatally, it is important to investigate them morphologically in order to elucidate the pathogenesis of the abnormalities. In this study, 13 45,X fetuses with cervical cystic hygroma were examined between 12 and 23 weeks of pregnancy. Every case had a hypoplastic thymus. The aortic valve was bicuspid in 11 cases and unicuspid in 2 cases. The aortic arch showed tubular hypoplasia between the left carotid artery and the left subclavian artery in 12 cases and type B interruption in one case. Smooth muscle cells and elastic fibers were reduced in number in the hypoplastic aortic arch. These results suggest hypoplastic development of the fourth branchial arch. Combined abnormalities between the aortic arch and aortic valve are not infrequently observed in DiGeorge anomaly. A similar developmental mechanism apparently underlies the pathogenesis of 45,X embryos. Possible genes causing the abnormalities are discussed.

A rare case of primary pulmonary choriocarcinoma in a male: immunohistochemical detection for human chorionic gonadotropin, epidermal growth factor (EGF) and EGF-receptor.

a rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.

Absent aortic and pulmonary valves: investigation of three fetal cases with cystic hygroma and review of the literature.

Absent semilunar valve was found in three fetal cases with cystic hygroma. Two cases which simultaneously showed absent aortic and pulmonary valves (AAV and APV, respectively) had double-outlet right ventricle. The third case, which lacked only the aortic valve, had atrioventricular septal defect and anomalous origin of the right subclavian artery. Two of the three cases had a markedly hypoplastic thymus. Fifteen AAV and 179 APV cases, including the above-mentioned cases and others reported elsewhere, were discussed with special reference to the pathogenesis of absent semilunar valve. Of the 15 AAV cases, hypoplasia of the left heart was observed in 11 cases (73.3%), double-outlet right ventricle in 5 (33.3%), and aortic arch malformations in 6 (40.0%). In the 179 APV cases, there were 111 tetralogy of Fallot (62.0%) and 44 right-sided aortic arch (24.6%). DiGeorge anomaly was found in one AAV and eight APV cases. These results indicate a pathogenesis that is possibly related to hemodynamic abnormalities or abnormal neural crest cells. Further investigation will be needed to elucidate a more definite pathogenesis of absent semilunar valve.

Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.

The purpose of this study is to reconstruct an alveolus-like structure from alveolar type II epithelial cells in a culture condition. Isolated alveolar type II epithelial cells of the rat were cultured in a three-dimensional collagen gel matrix. Single type II cells formed cellular aggregates that had a lumen after cell division in this culture condition. Through proliferation of the component cells, these aggregates grew to assume a globular or branching structure, part of which in turn developed into a large, cystic alveolus-like structure. This structure consisted of flattened epithelial cells intermingled with cuboidal epithelial cells. In these structures, the surfactant production was confirmed by immunohistochemistry and electron microscopy. To our knowledge, this is the first report of a reconstruction of an alveolus-like structure in a three-dimensional collagen gel matrix culture. This culture system seems to provide an appropriate physiological environment in which to study the differentiation and disorders of pulmonary alveoli.

Morphological and functional comparison of subcapsular small cells, subcapsular large cells and inner layer cells of bovine adrenal cortex.

Subcapsular small cortical cells (SC cells) and subcapsular large cortical cells (LC cells) of bovine adrenal cortex were cultured separately after purification by unit gravity sedimentation, and then compared with inner-half layer cells (IL cells) prepared by the same method. Both SC and LC cells were polygonal in shape and their mitochondria were elongated with lamellar cristae. SC cells became as large as LC cells on day 6 of culture with increased cytoplasmic lipid droplets, whereas IL cells showed no change in size. IL cells were spindle-shaped and had mitochondria with tubulovesicular cristae. Both SC and LC cells produced 11 beta-deoxycorticosterone, corticosterone, aldosterone and small amounts of 17 alpha-hydroxyprogesterone (17 alpha-OH-Prog) and cortisol (F). IL cells produced much more 17 alpha-OH-Prog and F than SC or LC cells. When stimulated with ACTH, cortical cells in each group showed cellular retraction and their mitochondria became spherical. The amounts of 17 alpha-OH-Prog and F increased in all groups, especially in IL cells. These results show that LC cells have similar characteristics to SC cells in both morphology and function, and that they differ from IL cells, which correspond to classical fasciculata-reticularis cells.

Setleis bitemporal "forceps marks" syndrome and its pathogenesis: a case report.

Setleis bitemporal "forceps marks" syndrome is characterized by "forceps marks" and a peculiar facies. The syndrome has previously been reported mainly in the Puerto Rican population. We describe here a Japanese boy with the syndrome. In addition, the hypothesis is presented that the Setleis syndrome may result from an insufficient migration of neural crest cells into the frontonasal process and the first branchial arch.

Reconstruction of the skin in three-dimensional collagen gel matrix culture.

The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The top-most layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.

The production of mouse fetal-placental chimeras using trisomy 16 and euploid blastocysts.

By means of a combination of immunosurgery and a modified method of microsurgery, blastocysts were reconstructed to produce viable chimeric fetal-placental units. Reciprocal reconstituted blastocysts were produced using euploid and trisomy 16 blastocysts. Reconstructed blastocysts yielded significantly smaller fetuses at day 17 of pregnancy than simultaneously transferred control blastocysts (mean body weight 0.49 g vs 0.64 g, P less than 0.01). However, apart from reduced size, no abnormalities were observed for any euploid fetus-euploid placenta construct. The three reconstructed blastocysts that yielded a trisomic fetus-trisomic placenta were viable when examined on day 17 and displayed the abnormalities typical of mouse trisomy 16. No reconstructed blastocyst that yielded a trisomic fetus-euploid placenta or a euploid fetus-trisomic placenta was viable beyond day 13 of development. One case in which a trisomic fetus had a placenta that was chimeric (euploid/trisomic) examined on day 17 displayed the abnormalities typical of a trisomic fetus but the placenta appeared histologically normal. The findings suggest that there is a coordination of the development of the fetus and the placenta that is essential for the development of the fetus.

Significance of cardiovascular malformations in cystic hygroma: a new interpretation of the pathogenesis.

Fetuses with cystic hygroma or loose skin of the neck were studied chromosomally and phenotypically to clarify the relation between neck abnormality and cardiovascular malformations. Of 12 fetuses, 9 had chromosome abnormalities: 4 with 45,X, 3 with trisomy 21, one each with trisomy 13, dup 6q. One had normal chromosomes. Two cases, in which chromosome analysis was unsuccessful, were morphologically suspected to be trisomy 13. Nine of the 12 fetuses had either bilateral cystic hygroma of the neck (7 cases) or nuchal bleb (2 cases: trisomy 13 and dup 6q). Two of the 3 remaining cases (trisomy 21) had loose skin of the neck, and one had edematous swelling of the skin of the neck. Except for the last case of trisomy 21, 11 fetuses (91.7%) had severe and/or rare cardiovascular malformations. They were divided into 3 major groups: a) spectrum of hypoplastic left heart syndrome (45,X and dup 6q), b) double outlet right ventricle, agenesis of semilunar valve (trisomy 13), and c) abnormality of atrioventricular orifice or valves (trisomy 21). One fetus with normal chromosomes had persistent left superior vena cava instead of absent right one and calcification of myocardium. Histological observation of edematous skin demonstrated the abnormal distribution of lymph vessels, including their absence. Some cases showed hypoplastic thymus. To integrate the findings of the present study and the descriptions in the literature, a pathogenesis is hypothesized in relation to migration of neural crest cells and extracellular matrix.

A simple culture method of fat cells from mature fat tissue fragments.

To obtain immature fat cells in vitro, we used a primary culture of undigested mature fat tissue fragments. The immature fat cells, i.e., fibroblast-like fat cells, proliferated extensively from the fat tissue and differentiated after reaching confluence. The process of differentiation was assumed by the development of intracytoplasmic lipid droplets and by the triglyceride content in the cells. Cellular differentiation was induced in high percentages (over 70-80%) of the cells in the medium containing high glucose concentrations (200 mg/dl) supplemented with 10-20% newborn calf serum. The intracellular accumulation of triglyceride was also enhanced by insulin administration. In these cells, a reciprocal relationship was observed between proliferation and differentiation. Fibroblast-like fat cells derived from mature fat tissue in this simple culture system are suitable for the study of the proliferation and differentiation of immature fat cells.

Polypoid squamous cell carcinoma of the larynx. An immunohistochemical study for ras p21 and cytokeratin.

Polypoid squamous cell carcinoma of the larynx consists of two components, namely, squamous cell carcinoma and sarcomatoid spindle cells. To further investigate the histogenesis of the spindle cell component, we studied two cases of polypoid squamous cell carcinoma of the larynx by immunohistochemistry and ultrastructural analysis. Positive staining for ras oncogene p21 and cytokeratin was demonstrated in both squamous cell carcinoma and spindle cell component. Only the latter component was positive for vimentin. Electron microscopic examination showed well-developed desmosomes in spindle cells. These results suggest that the spindle cell component is epithelial in origin and malignantly neoplastic; it originates from mesenchymal metaplasia or squamous cell carcinoma.

[A case of cystic changes in a giant leiomyoblastoma of the stomach].

A 44-year-old male was admitted to hospital with a complaint of abdominal distention. A CT scan revealed a giant multicystic mass in the abdominal cavity, a tumor that measured 34 cm in diameter, which had risen from the gastric body and grew in an exophytic fashion with a stalk. Histologic findings exhibited a malignant leiomyoblastoma with cellular pleomorphism and a intracytoplasmic cystic change, which included disseminated nests of immature small round cells. After excisional surgery, the patient has had no sign of a recurrence for the past two years.

Testicular tumors in non-twin brothers from a consanguineous marriage.

Malignant testicular tumors occurring in non-twin brothers are reported. Both of the brothers suffered from amentia and epilepsy and were the product of a consanguineous marriage. One brother presented with teratocarcinoma and the other seminoma. With a review of the literature, genetic roles in the etiology of testicular neoplasia are discussed.

Unilocular fat cells in three-dimensional collagen gel matrix culture.

Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture.