Development and characterization of primary cultures of smooth muscle cells from the fibromuscular stroma of the guinea pig prostate

Summary Primary cultures of smooth muscle cells (SMCs) were obtained by a two-step enzymatic digestion of guinea pig prostatic stroma. Ultrastructural morphology and growth characteristics of these cells conformed to those reported for SMCs isolated from vascular and visceral tissue sources. Electron microscopic examination indicated that the cells assumed modified myofibroblastoid features in culture. Microfilaments with associated dense bodies were markedly depleted in cultured smooth muscle cells, in comparison with those of the parent tissue. Cultured cells also possessed increased content of rough endoplasmic reticulum indicating the increased secretory or protein-synthetic capacity of the cells. Immunoperoxidase staining for cytoskeletal markers using monoclonal antibodies to desmin and vimentin supported the ultrastructural observations, suggesting a decline in desmin-staining intermediate filaments during “modulation” to the myofibroblastoid form. Despite this depletion of smooth muscle-specific differentiation markers and reversion to more general mesenchymal properties, the cells retained the ability to contract on challenge with norepinephrine, and grew in the characteristic “hill and valley” pattern on attaining confluence. Inasmuch as the estrogen and androgen receptor expression of the parent stromal tissue is also retained, these primary cell cultures should provide a useful model to study regulation of prostatic development. This work was supported by research grants from the National Health and Medical Research Council of Australia, the Anti Cancer Foundation of the Universities of South Australia, and the Flinders Medical Centre Research Foundation.     

Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance

We have isolated and characterized the gene encoding the human androgen receptor. The coding sequence is divided into eight coding exons and spans a minimum of 54 kilobases. The positions of the exon boundaries are highly conserved when compared to the location of the exon boundaries of the chicken progesterone and human estrogen receptor genes. Definition of the intron/exon boundaries has permitted the synthesis of specific oligonucleotides for use in the amplification of segments of the androgen receptor gene from samples of total genomic DNA. This technique allows the analysis of all segments of the androgen receptor gene except a small region of exon 1 that encodes the glycine homopolymeric segment. Using these methods we have analyzed samples of DNA prepared from a patient with complete androgen resistance and have detected a single nucleotide substitution at nucleotide 1924 in exon 3 of the androgen receptor gene that results in the conversion of a lysine codon into a premature termination codon at amino acid position 588. The introduction of a termination codon into the sequence of the normal androgen receptor cDNA at this position leads to a decrease in the amount of mRNA encoding the human androgen receptor and the synthesis of a truncated receptor protein that is unable to bind ligand and is unable to activate the long terminal repeat of the mouse mammary tumor virus in cotransfection assays.     

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua,New Guinea

Summary Melanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta 1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.     

Intrinsic electric fields and membrane bending

The fragmentation of human erythrocytes heated in a range of ionic environments has been examined by video microscopy, , the average number of surface wave crests growing on the cell rim during fragmentation by membrane externalization, andI, the percentage of cells internalizing membrane, were scored.The membrane diffusion potential was altered experimentally on decreasing the extracellular chloride concentration by substituting either membrane-impermeant sorbitol or Na gluconate for some NaCl. The external-membrane-face surface potential was altered either by surface charge depletion or by ionic strength changes. The dependence of morphological change on diffusion potential at constant cell volume and surface potentials was established over a 34-mV change in diffusion potential. The rate constants for morphological change with charge depletion at different diffusion potentials are largely independent of the diffusion potential. A l.O-mV increase in diffusion potential has an effect on morphological change of comparable magnitude to that of a 1.0-mV decrease in the modulus of the negative surface potential. When the diffusion potential increased on decreasing both the extracellular diffusible ion concentration and extracellular ionic strength, the effect on cell morphology of increasing the modulus of the surface potential was overcome by the effects of the diffusion potential change.     

Minireview: The androgen receptor in breast tissues: growth inhibitor, tumor suppressor, oncogene?

Androgen receptor (AR) signaling exerts an antiestrogenic, growth-inhibitory influence in normal breast tissue, and this role may be sustained in estrogen receptor α (ERα)-positive luminal breast cancers. Conversely, AR signaling may promote growth of a subset of ERα-negative, AR-positive breast cancers with a molecular apocrine phenotype. Understanding the molecular mechanisms whereby androgens can elicit distinct gene expression programs and opposing proliferative responses in these two breast cancer phenotypes is critical to the development of new therapeutic strategies to target the AR in breast cancer.