👤 Atsuto Onoda

A gain-of-function mutation in germline ABL1 causes a syndrome including congenital heart defects. However, the molecular mechanisms of this syndrome remain unknown. In this study, we found a novel ABL1 mutation in a Japanese family with ventricular septal defect, finger contracture, skin abnormalities and failure to thrive, and the molecular mechanisms of these phenotypes were investigated. Whole-exome sequencing on several family members revealed a novel mutation (c.1522A > C, p.I508L) in the tyrosine kinase domain of ABL1, and complete co-segregation with clinical presentations was confirmed in all members. Wild-type and mutant ABL1 were transfected into human embryonic kidney 293 cells for functional analysis. Western blotting confirmed that tyrosine phosphorylation in STAT5, a substrate of ABL1, was enhanced, and the novel mutation was proved to be a gain-of-function mutation. Since this novel mutation in ABL1 enhances tyrosine kinase activity, phosphorylated proteome analysis was used to elucidate the molecular pathology. The proteome analysis showed that phosphorylation in proteins such as UFD1, AXIN1, ATRX, which may be involved in the phenotypes, was enhanced in the mutant group. The onset of congenital heart defects associated with this syndrome appears to involve a mechanism caused by UFD1 common to 22q.11.2 deletion syndrome. On the other hand, AXIN1 and ATRX may be important in elucidating the mechanisms of other phenotypes, such as finger contracture and failure to thrive. Verification of these hypotheses would lead to further understanding of the pathophysiology and the development of treatment methods. Show less

The expression of guanine nucleotide-binding proteins (G proteins) during the development of rat testes was investigated. Immunohistochemical studies on frozen sections and isolated testicular cells demonstrated that the expression of the GTP-binding proteins was developmentally regulated and specific for different cell types. The alpha subunit of the cholera toxin-sensitive stimulatory G protein (Gs alpha) was first detected in testes from 7-day-old rats; its value reached a maximum at 23 days and then decreased to very low or undetectable amounts in testes of 45-day-old and adult rats (60-90 days of age). The Gs alpha subunit appears to be expressed by Sertoli, peritubular myoid and interstitial cells. The common beta subunit (G beta) was present at all ages during development and was more prominent around the periphery of the tubules in younger animals but then became more evident in the cytoplasm of germ cells with increasing age. The pertussis toxin-sensitive inhibitory G proteins, Gi1/2 and Gi3, showed a similar pattern of expression. Sertoli cells and peritubular cells expressed Gi1/2 and Gi3 at very low levels at all ages, whereas pachytene spermatocytes and round spermatids expressed the inhibitory binding proteins only at later ages of development (45-day-old and adult testis). Northern blot analysis showed that with increasing age the Gs alpha mRNA in the testis decreased and this was confirmed by in situ hybridization. These latter studies showed localization of the transcripts to somatic cells but not to germ cells.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Sertoli cells from immature rats (18 days old) were cultured on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Three types of cultures were obtained: 1) confluent monolayer cultures that formed a permeability barrier (impermeable), 2) confluent monolayer cultures that did not form a permeability barrier (permeable), and 3) subconfluent cultures (permeable). The relationships among fluid equilibrium, electrical resistance, and [3H]inulin transport between the apical and basal reservoirs of the chambers were examined. An impermeable confluent monolayer is defined when the cells of the Sertoli cell epithelial sheet are able to prevent hydrodynamic equilibration of fluid levels between the apical and basal reservoirs of a bicameral chamber. That is, a permeability barrier is present between the two sides of the chamber when fluid levels (volumes) do not change. In the impermeable confluent Sertoli cell monolayers, 7.5 +/- 0.6% of added [3H]inulin diffused across the monolayer during a 6-h collection period versus 13.7 +/- 0.5% in permeable cultures. Conversely, the electrical resistance was higher in the impermeable monolayers (41-71 ohm.cm2) than in the permeable layers (less than 33 ohm.cm2). A reciprocal linear relationship (Y = -4.68(X) + 91.50, r = 0.808) exists between inulin flux and electrical resistance, and this relationship is a function of cell density. Transferrin (Tf) was one of a few proteins detected in the basal medium of bicameral chambers, whereas most de novo synthesized proteins were secreted into the apical reservoir of the chamber. No significant differences in the total amount of Tf secreted by impermeable or permeable monolayers of Sertoli cells were observed. However, the Sertoli cell secretion ratios (apical/basal) of Tf during a 15-20-h collection period were 2.03 and 1.57 for impermeable monolayers plated at 2.4 x 10(6) and 3.6 x 10(6) cells/well, respectively, but less than 1.0 in permeable layers of cells. When fewer than 2 x 10(6) Sertoli cells were plated, the apical/basal polarity of Tf secretion declined to below 1 in a 24-h culture period, even though those chambers contained impermeable monolayers (recognized by the lack of hydrodynamic equilibrium). These results indicate that polarized secretion by Sertoli cells is dependent on (1) plating density and (2) formation of an impermeable epithelial sheet. Show less

Epidermal growth factors receptor (EGFR) was localized immunocytochemically in the testes of mature and immature rats and immature monkeys. One polyclonal antibody, recognizing the intracellular domain (RK2) of the receptor, was used to carry out the EGFR immunodetection. The RK2 antibody revealed the presence of the EGFR predominantly in Sertoli cells of mature and immature rats and of immature monkeys, although limited interstitial localization of the EGFR was also discerned in the mature rat. In cultured Sertoli cells of immature rats, grown in the absence of epidermal growth factor (EGF), the EGFR was randomly distributed at the cell surface, whereas after the addition of EGF the receptor became aggregated into distinct focal regions. In addition, EGFR of cultured Sertoli cells exhibited autophosphorylation activity upon stimulation with EGF, but failed to transcytose iodinated EGF across a permeability barrier formed by the cultured cells. Instead, all of the added iodinated EGF was internalized and degraded. Show less