Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

Effects of polyamines and histone on the phosphorylation of non-histone proteins in isolated rat liver nuclei

Addition of polyamines to isolated nuclei increases the rate and extent of phosphate incorporation from ATP into non-histone proteins several-fold. Similar results are obtained when histones are added to phosphorylating nuclei or when nuclei are incubated with DNAase prior to the addition of ATP. Electrophoretic analysis of the reaction products in SDS polyacrylamide gels reveals that specific non-histone proteins are preferentially phosphorylated in the presence of polyamines, some of which appear to be the same as in the presence of histones or DNAase. Removal of protein-bound phosphate during prolonged incubation of nuclei occurs with the same kinetics in the presence or absence of polyamines. Our results suggest that polyamines and histones stimulate nuclear protein phosphorylation by rendering additional phosphate acceptors accessible to the kinases.     

Differential conjugation of polyamines to calf nuclear and nucleolar proteins

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Analyses of ribosomal proteins from mycobacteria with particular reference to the β antigen

Abstract The proteins of the ribosomal subunits of Mycobacterium phlei were analysed by sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by 2-dimensional PAGE. These techniques revealed that both the size and the charge of the mycobacterial ribosomal proteins were quite different from those of Escherichia coli . The divergent chemical properties of the mycobacterial ribosomes might be related to other exceptional properties of mycobacteria, e.g., their slow growth. An antigen designated β was furthermore revealed in both 30S and 50S subunits of M. phlei and Mycobacterium bovis BCG. 2 Proteins from each subunit migrated in a similar way in SDS-PAGE, being thus prime candidates as carriers of β-activity.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

DNA and ribonucleotide binding characteristics of two forms of the androgen receptor from rat prostates

Androgen receptors (sedimentation value approximately 4S and Stokes radius 2.8 nm) present in the cytoplasmic fraction obtained from prostates of castrated rats bind to DNA-Sepharose and double stranded DNA. A receptor fragment (sedimentation value approximately 3S and Stokes radius 2.3 nm) obtained from rat prostates in the course of a purification procedure showed greatly diminished binding affinity for both DNA-Sepharose and soluble DNA. In contrast, both the 4S cytosol receptor and the 3S receptor form interacted with equal affinity with prostate RNA or poly(UG). These observations provide evidence that for DNA binding a different or additional part of the receptor molecule is required than for RNA and polyribonucleotide binding.     

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates

Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca⁺⁺-and Mg⁺⁺-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis. This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from the National Cancer Institute, National Institutes of Health, Bethesda, MD.     

Effect of polyamines on phosphorylation of non-histone chromatin proteins from hog liver.

The effects of polyamines on the $\text{in vitro}$ phosphorylation of non-histone chromatin proteins from hog liver has been found to be dose dependent. Maximal increase occurred at 0.2 mM spermine and 2 mM spermidine, respectively. These results suggest that spermine and spermidine may have a regulating function for phosphorylation of non-histone chromatin proteins in hog liver.     

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

Axoplasmic proteins of the squid giant nerve fiber with particular reference to the fibrous protein

1. Axoplasm of squid giant nerve fibers is examined with the ultracentrifuge and electrophoresis apparatus and several distinct components demonstrated. 2. One of these components, a protein called axon filaments, is isolated by fractional extraction followed by differential ultracentrifugation and redissolving in glycine solution. Axon filaments are monodisperse by ultracentrifugation. Their physical chemical properties have been studied. 3. The existence of a reversible transformation of axon filaments into a particle of lower molecular weight and lower asymmetry has been demonstrated.     

Effects of stent stiffness on local haemodynamics with particular reference to wave reflections

The placement of a rigid stent within an elastic vessel produces wave reflection sites at the entrance to and exit from the stent. The net haemodynamic effects of these reflections depend critically on the degree of stiffness of the stent and on its length and position within the diseased vessel, variables that have been found to affect the clinical performance of a stent. Here these effects are examined analytically, using a segmented tube model. The results indicate that the presence of the stent within the larger diseased vessel has the effect of producing higher pressure at the vessel entrance than that at exit. This pressure difference, when superimposed on the underlying pressure distribution within the vessel, has the net effect of actually aiding rather than impeding the flow, but the extent of this depends on the length and position of the stent. A short stent placed near the entrance of the diseased vessel may be favoured clinically for producing the least perturbation in the underlying haemodynamics and thus reducing the chance of restenosis, while a long stent placed near the exit may be favoured for producing a positive pressure difference and thus aiding the flow.     

A comparison of nuclear and nucleolar matrix proteins from rat liver

The comparison of the gel electrophoresis patterns of nuclear and nucleolar matrix proteins reveals marked differences between these structures. The nucleolar matrix contains 5 prominent protein bands ( mol.wt.: 8.2; 7.0; 5.6; 4.0 and 3.0 x 10 4 ) which are not found in nuclei. It is suggested that the nucleolar matrix has a distinct structure participating in selective interactions.     

Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate

Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms (Ka = 0.5-2.9 x 10(10) M-1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates.