Effect of Sodium Molybdate on Androgen Binding to Its Receptor from Shionogi Carcinoma 115

The effects of molybdate on the binding characteristics of androgen receptor from the androgen-dependent mouse tumor (Shionogi carcinoma 115) with 17/β-hydroxy-5α-androstan-3-one were studied by the charcoal adsorption method. The dissociation constant (Kd) was (4.65±0.54) ×1O-11 M in the presence of 10-12 mM sodium molybdate, which was one order of magnitude lower than the value without molybdate ((4.54±0.72)× 10-10 M). Since the half life of the binding activity of unbound receptor was extended from 10 h to 75 h by adding molybdate, the effects of chaotropic reagents on binding were studied in the presence of molybdate. KC1 (0.3 -1.0 M) remarkably increased Kd without affecting the maximum binding capacity or the dissociation rate. KSCN (>0.1 M) had no effect on Kd but increased the dissociation rate, and while at concentrations at or above 0.5 M it completely inhibited the androgen binding to the cytosol receptor. Urea (0.5-2.0 M) increased Kd proportionally to the increase in the dissociation rate. Kd was also increased by 0.2-0.5 M guanidine, but the maximum binding capacity was reduced

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

AbstractIn the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb

Genistein affects osteoblastic MC3T3-E1 cells both through estrogen receptor and BMP-Smad signaling pathways

Many epidemiological studies show that genistein intake is effective for maintaining bone mineral density (BMD). Because the reason for the efficacy of genistein as a bone protective agent in vivo remains unclear, we investigated the mechanisms underlying the effects of genistein on BMD in relation to BMP-Smad signaling systems. When osteoblastic MC3T3-E1 cells were exposed to 1 μM genistein, they increased in number. Combined administrations of 1 μM genistein and 1 μM of ICI 182,780 inhibited the increase in cell numbers. Alkaline phosphatase (ALP) and Alizarin red staining showed high activities, indicating that genistein might promote estrogenic differentiation of MC3T3-E1 cells. Moreover, ELISA determined that production of osteoprotegerin (OPG), which is expressed by osteoblasts, was higher when 1 μM genistein was added to the medium than in controls. In contrast, when 10 ng/mL of noggin was administered in the medium, OPG production was inhibited. In order to clarify the underlying mechanism, we investigated the BMP-Smad signaling pathway. When genistein was added to the medium, it induced gene expression of BMP-4. Immunofluorescence staining showed that genistein induced phosphorylation of Smad 1/5, a downstream molecule of BMP. When noggin, which binds to BMP and blocks BMP signaling, was added to the medium, phosphorylation of Smad 1/5 was reduced. These results indicate that genistein may regulate bone metabolism through the BMP-Smad signaling pathway as well as through the estrogen receptor pathway

Spontaneous glomerular deposition of immunoglobulins for ACE (Angiotensin Converting Enzyme) induces spontaneous nephropathy in diabetogenic rats

We first discovered autoantibodies to ACE (angiotensin converting enzyme) in patients with type 2 diabetes mellitus^1^. The antibodies were positive for 64.5% of the patients with diabetes and were positive in 83.3% in the early stage of clinical diabetic nephropathy. In addition, in genetically diabetogenic OLETF (Otsuka Long-Evans Tokushima Fatty) rats^2^, one of the characteristics of which strain is spontaneous nephropathy resembling those of human type 2 diabetes, and in control LETO rats^2^, immunization with rabbit lung ACE developed glomerulopathy similar to that seen in diabetics^3^. Also, in normal New Zealand white rabbits, immunization with the rabbit lung ACE induced glomerular changes similar to those seen in diabetic nephropathy^3^. In this study, renal tissues identical to those examined in research of diabetic nephropathy by PAS staining and electron microscope in preceding study^3^, were examined by immunostaining methods, only to prove that the diabetic glomerular changes may occur by immunization with ACE, not by non-specific responses to ACE, in non-diabetogenic rats and rabbits

Glycosylphosphatidylinositol-Anchored Cell Surface Proteins Regulate Position-Specific Cell Affinity in the Limb Bud

AbstractAlthough regional differences in mesenchymal cell affinity in the limb bud represent positional identity, the molecular basis for cell affinity is poorly understood. We found that treatment of the cell surface with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) could change cell affinity in culture. When PI-PLC was added to the culture medium, segregation of the progress zone (PZ) cells from different stage limb buds was inhibited. Similarly, sorting out of the cells from different positions along the proximodistal (PD) axis of the same stage limb buds was disturbed. Since PI-PLC can remove glycosylphosphatidylinositol (GPI)-anchored membrane bound proteins from the cell surface, the GPI-anchored cell surface proteins may be involved in sorting out. To define the GPI-anchored molecules that determine the segregation of limb mesenchymal cells, we examined the effect of neutralizing antibody on the EphA4 receptor that binds to GPI-anchored cell surface ligands, called ephrin-A. Sorting out of the PZ cells at different stages could be inhibited by the neutralizing antibody to EphA4. These results suggest that EphA4 and its GPI-anchored ligands are, at least in part, involved in sorting out of limb mesenchymal cells with different proximal–distal positional values, and that GPI-anchored cell surface proteins play important roles in determining cell affinity in the limb bud

Growth Characteristics of KB and RAJI Cells in the Liquid Culture Medium in Relation to the Human Tumor Stem Cell Assay for Anti-Tumor Agents

Recently the chemosensitivity assay with clonal human tumor stem cells in liquid medium have been developed. We have investigated the optimal conditions for culture and counting of KB cells, which derived from a human epidermoid carcinoma and grow as a sheet adhered to the bottom surface of the culture, and RAJI cells, which derived from Burkitt lymphoma and grow as a suspension in liquid medium, as the representatives of the human tumor cells. Both stock cells were maintained in Eagle\u27s MEM medium plus 10 per cent calf serum in C02 incubator at 37°C. Trypsinized cell suspensions were delivered in plastic wells with different culture media and incubated for different periods. Then the cell number was counted by the Coulter Counter in ISOTON II solvent. For KB cells, the culture medium should not be changed for 72 hours, otherwise enough cell number could not be obtained. To prevent the aggregation of KB cells in ISOTON II, calf serum (10%) was effective and EDTA (0.02%) had an additive effect. RAJI cells did not aggregate in ISOTON II. Ethanol and dimethylsulfoxide inhibited the growth of RAJI cells, but almost no inhibition was noted when the initial cell number was more than 8×l04/ml. Ethanol was more toxic. Both solvents, however, did not affect the growth of KB cells

Molecular Cloning of the Escherichia coli Glutamine Permease Operon, glnP, Using Mini-F Plasmid

The genes encoding the Escherichia coli high-affinity transport system for glutamine, glutamine permease operon, were cloned by means of phenotypic complementation using a mini-F cloning vehicle. Plasmid pTN101 contains the structural gene for the periplasmic glutamine binding protein, glnH, and at least one other gene, glnP, also required for the permease activity. It is difficult to reclone the entire operon into high-copy number plasmids pBR322 or pUC18: The resultant plasmids are extremely unstable and are rapidly lost from the host cell. Subcloning of the operon in pBR322 resulted in the loss of Gln+ phenotype, an ability to utilize glutamine as a sole source of carbon. Direct cloning of the permease operon by using pBR322 was unsuccessful; although three types of Gln+ growers were obtained, they do not contain DNA in the glnP locus