Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.     

Epoxy ceriporic acid produced by selective lignin-degrading fungus Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white rot basidiomycete which degrades lignin in wood at a distance far from enzymes. Low molecular mass metabolites play a central role in the oxidative degradation of lignin. To understand the unique wood-decaying mechanism, we surveyed the oxidized derivatives of ceriporic acids (alk(en)ylitaconic acids) produced by C. subvermispora using high-resolution liquid chromatography multiple-stage mass spectrometry (HR-LC/MSⁿ). The analysis of the precursor and product ions from the extract suggested that an epoxidized derivative of ceriporic acid is produced by the fungus. To identify the new metabolite, an authentic compound of ceriporic acid epoxide was synthesized in vitro by reacting (R)-3-[(Z)-hexadec-7-enyl]-itaconic acid (ceriporic acid C) with m-chloroperbenzoic acid. The precursor and product ions from the natural metabolite and authentic epoxide were identical and distinguishable from those of hydroxy and hydroperoxy derivatives after reduction with NaBD₄. Feeding experiments with [U-¹³C]-glucose, 99% and the subsequent analyses of the first and second generation product ions demonstrated that the oxidized ceriporic acid was (R)-3-(7,8-epoxy-hexadecyl)-itaconic acid. To our knowledge, this study is the first to report that natural alkylitaconic acid bears an epoxy group on its side chain.     

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.     

Timing and sequence of the events in the development of extraocular muscles in staged human embryos: ultrastructural and histochemical study.

The ultrastructure and the appearance of glycogen were studied in the extraocular muscles of 14 externally normal human embryos (Carnegie stages 13-21). At stage 16, myofibrils with an immature Z line and glycogen granules appeared in the cytoplasm of the myoblast. The myoblasts came into cluster at stage 18, and fusion between the myotubes was observed at stage 20. At this stage, an M line appeared in the myofibrils. At stage 21, an A band with a Z line and an H band with an M line were observed, the sarcoplasmic reticulum appeared in the cytoplasm of the muscle fibers and glycogen increased in volume in the cytoplasm. In the previous study, we showed that the muscle-specific isoenzymes, such as creatine kinase, beta-enolase and glycogen phosphorylase, appeared from stage 18 to 20 in the extraocular muscles. The previous findings and the present results suggest that the fusion of the muscle cells occurs in the period when some molecular markers of muscle differentiation are expressed in vivo.     

Purification of a serum DNA binding protein (64DP) with a molecular weight of 64,000 and its diagnostic significance in malignant diseases

A DNA binding protein with a molecular weight of 64,000(64DP) has been purified to homogeneity from human serum, and its quantitative assay has been developed. The average level of serum 64DP in 30 normal controls was 41.4 μg/ml, whereas it was 175 μg/ml in 87 patients with untreated malignant disease. Furthermore it was found to be elevated in all tested patients, 8 cases, with carcinoma in early stages. Serum 64DP has been found to be different from C3DP, CEA^# or α-FP^#, and it appears that this protein might prove to be a useful tumor marker in malignant diseases.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.     

Ultrastructural study on the retinal pigment epithelium of human embryos, with special reference to quantitative study on the development of melanin granules

The development of the retinal pigment epithelium (RPE) was studied ultrastructurally, using 13 externally normal human embryos, Carnegie stages ranging from 13 to 23 (4-8 week of gestation). Melanosomes in the peripheral and posterior RPE were classified according to Fitzpatrick et al. The melanosome of phase I is formed from the Golgi complex and parcelled off into small vesicles. The vesicle enlarges and elongates to form an oval organelle with membranous structures in it (phase II melanosome). Subsequently, melanin deposits on the membranous structures of the melanosomes (phase III melanosomes), and the completion of this process produces a uniformly electrondense granule without discernible internal structures (phase IV melanosome). Melanosomes of phases III and IV appeared in the RPE at stage 15. As the embryonic stage advanced, the ratio of phase II melanosomes decreased and that of phase IV melanosomes increased. The number of phase III melanosomes reached a peak in the peripheral and posterior RPE at stages 15 and 18, respectively. After stage 17, the increase in melanosomes and intracellular organelles was more prominent in the posterior than in the peripheral RPE. During stages 13 and 15, gap junctions were present not only in the apical but also basal plasma membranes of the RPE. At stage 20, gap junctions in the basal plasma membrane disappeared except for the transitional areas from the RPE to the neural retina (NR). In addition, gap junctions were observed between NR and RPE only in the peripheral region at stage 20. The morphological and quantitative differences in the peripheral and posterior RPE in the embryonic period are discussed.     

The tumor suppressor LKB1 induces p21 expression in collaboration with LMO4, GATA-6, and Ldb1

The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers syndrome and various sporadic cancers such as lung adenocarcinoma. We show here that LKB1 forms a complex with LMO4, GATA-6, and Ldb1, and enhances GATA-mediated transactivation in a kinase-dependent manner. We further demonstrate that LKB1 has the potential to induce p21 expression in collaboration with LMO4, GATA-6, and Ldb1 through the p53-independent mechanism. Our findings suggest that LKB1 regulates GATA-mediated gene expression and that this activity of LKB1 may be important for its tumor suppressor function.     

Ontogeny of GTP-binding proteins,Gi and G0, in rat retina

The distribution and the levels of G_i1 (plus G_i3), G_i2, and G₀ in rat retina were studied immunohistochemically and immunochemically during development. At embryonic day (E) 15, G_i1α/G_i3α was observed in the inner layer of the neural retina, the future nerve fiber layer (NFL), while G_i2α was observed both in the inner and outer layers of the neural retina. No immunoreactivity for G₀α was observed. At E18, G_i1α/G_i3α and G_i2α appeared in the inner plexiform layer (IPL), while G₀α was faintly immunoreactive only in the NFL. At birth, G_i2α/G_i3α and G₀α appeared in the ganglion cell layer. G_i2α was intensely immunoreactive in the NFL and IPL. At postnatal day (P) 10, the inner portions of the retina, from the NFL to the outer plexiform layer, were immunoreactive to G_i1α/G_i3α, G_i2α, and G₀α. G_i1α/G_i3α and G₀α were distributed characteristically in a laminated pattern in the IPL, but G_i2α was present homogeneously in the IPL. At P12, G_i2α appeared in the outer nuclear layer. As the postnatal days advanced, the laminated pattern of immunoreactivity to G₀α in the IPL became diffuse, but immunoreactivity to G_i1α/G_i3α remained. The results of enzyme immunoassays showed that the concentration of G₀α increased rapidly from P10 to P15 and reached almost the adult level at P20–P30, while G_i2α decreased until P15 and was almost constant thereafter. These results showed that the distribution of G_i1α/G_i3α, G_i2α, and G₀α differs during development, suggesting that each G protein in the developing retina has a unique function.