The effect of age on the response of the detrusor to intracellular mechanical stimulus: DNA replication and the cell actin matrix.

Benign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myocyte ultrastructure and consequent effects these might have in modifying the response of the detrusor to mechanical stimulus, we studied differences in dynamic nuclear-cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which specifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2-3 week) and old (8-12 mon) guinea pigs. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracellular stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti-alpha-smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor myocytes showed dose dependent disruption of the actin component of the cytoskeleton. These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the detrusor in response to outflow obstruction in infancy and adulthood. Furthermore, they suggest that 1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; 2) there appears to be a decrease in myocyte nuclear elasticity with ageing; 3) release of nuclear template restrictions increases activity of DNA polymerase alpha in young, but not old, detrusor myocytes; and 4) mechanico-chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that 1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and 2) integrity of nuclear actin may function to maintain the spatial template restriction on DNA polymerase alpha activity.     

Collagen increases the synthesis of membrane-associated proteoglycans produced by Sertoli cells.

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium.     

Effects of the stereochemical orientation of phenethylamines and imidazolines on alpha-adrenergic receptor-mediated DNA synthesis in primary cultured rat hepatocytes.

The hepatic alpha 1-adrenergic receptor mediates a variety of hepatic functions including respiration, glycogenolysis, gluconeogenesis, and growth. We have utilized a rat primary hepatocyte culture system to show that the alpha 1-adrenergic receptor can be activated in a stereoselective manner by a series of phenethylamines and catecholimidazolines resulting in the stimulation of DNA synthesis as determined by [3H]thymidine incorporation. The phenethylamines adhered to the Easson-Stedman hypothesis with a rank order of potency of (-)-(R)-norepinephrine (NE) greater than (+)-(S)-NE greater than the desoxy analog dopamine (DA) for the stimulation of DNA synthesis. However, the 2-substituted catecholimidazolines did not follow this trend and demonstrated an order of potency of the desoxy analog 3,4-dihydroxybenzyl imidazoline (DHT) greater than or equal to (-)-(R)-2-(3,4,alpha-trihydroxybenzyl)imidazoline (TBI) greater than (+)-(S)-TBI. 4-Substituted catecholimidazolines were less potent as inducers of DNA synthesis than the corresponding 2-substituted analogs with an order of potency of (+)-(R)-4-(3,4-dihydroxybenzyl)imidazoline (DBI) greater than (+,-)-(R,S)-DBI greater than (-)-(S)-DBI. When the beta-hydroxyl moiety of NE is replaced with an amino group as in 3,4-dihydroxyphenylethylenediamine, the isomers are less active than the beta-hydroxylated analogs and also demonstrate no stereoselectivity for the stimulation of DNA synthesis. These results demonstrate that the hepatic alpha 1-adrenergic receptor can recognize various isomeric forms of these compounds and that hepatocellular growth can be modulated in a stereoselective manner by phenethylamines and imidazolines.     

Genetic assay of misincorporation

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.