A novel nuclear protein in rat ventral prostate androgen-dependent and age-related change

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to $\text{1}\text{60}$ of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H₁ and H₁⁰ histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO₄, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or brain, suggesting that it is specific to the ventral prostate.     

Nuclear non-histone proteins from rat ventral prostate cell under going hypertrophy of hyperplasia.

The distribution of nuclear non-histone proteins and their labelling with [¹⁴C]amino acids or [³²P]O₄ in rat ventral prostate cells undergoing hypertrophy (cell growth) or hyperplasia (cell division) were compared by SDS polyacrylamide gel electrophoresis. Marked quantitative changes in these proteins accompanied castration and replacement of hormone, but no absolute qualitative differences in their staining patterns were established. Preparations from cells undergoing hyperplasia were augmented in high molecular weight proteins and contained fewer proteins of <20000 D. The increased amounts of nuclear non-histone proteins from hormone-treated castrated rats were highly labelled by [¹⁴C]amino acids, but the molecular weights of radioactive proteins from cells undergoing hypertrophy were less diverse than those from replicating cells, Phosphorylation of nuclear non-histone proteins from short-term and long-term castrates, treated with testosterone propionate, was 170 and 60% greater than their controls. Proteins from 20–45 × 10³ D were actively phosphorylated. Nuclear extracts from dividing cells contained additional radioactive high molecular weight proteins and fewer phosphorylated lower molecular weight components. The distribution of phosphorylated proteins and newly synthesized proteins was dissimilar. Quantitative and possible qualitative differences in staining of nuclear proteins isolated in N-ethylmalleamideurea-phosphate buffer from normal or hormone-treated castrated rats were accentuated when they were separated by charge at pH 2.8. In replicating cells, a more generalized synthesis of acidic nuclear proteins from all molecular weight classes occurred, which were not as highly phosphorylated as less heterogeneous nuclear proteins from cells undergoing hypertrophy. Examination of the immediate and the subsequent events following androgen-induced cellular hypertrophy or hyperplasia in the ventral prostate permits their comparison in the same tissue.     

Ionophore A23187 raises cyclic AMP levels in macrophages by stimulating prostaglandin E formation.

A class of non-histone chromatin proteins that were bound tightly to DNA and could not be dissociated from the chromatin by high salt and urea was isolated from HeLa cell nuclei and separated from DNA by DNase digestion. These ‘tight’ proteins retained their ability to bind to single- and double-stranded DNA as assayed by nitrocellulose filter binding. Polyacrylamide gel electrophoresis showed that the most prominent proteins possessed molecular weights of about 60 000 D. In asynchronously growing HeLa cell cultures about $\text{1}\text{3}$ of the cell nuclei were immunoreactive to fluorescein-labeled antinucleoside antibodies. The immunoreactive cells were the fraction in S phase. Cycloheximide treatment of the cultures raised the fraction of immunoreactive nuclei to over $\text{sol2}\text{3}$. Exposure of the fixed cycloheximide-treated cell to tight proteins prior to staining with the antibody reduced the fraction of immunoreactive cells to the normal S phase level. Immuno-reactivity induced by X-irradiation or by the intercalating mutagen hycanthone was also suppressed by tight proteins. Cycloheximide treatment preferentially reduced the cellular content of tight proteins, suggesting that these proteins undergo a metabolic turnover with a half-life of about 5 h.     

Chronic treatment with valium (diazepam) fails to affect the reproductive system of the male rat

Male rats treated chronically with high doses of Valium (50mg/ Kg/day; 10 days) failed to exhibit changes in their reproductive system. Testicular and prostate weights, serum testosterone (T) and LH were unaffected. Testes and pituitary tissue stimulated $\text{in}$$\text{vitro}$ with LH and GnRH, respectively, released normal amounts of T, LH and FSH. Brain benzodiazepine receptors were slightly but significantly elevated by Valium treatment as well as by castration. We conclude that the male reproductive system is resistant to chronic Valium treatment even though the brain levels of benzodiazepine receptors are not.     

Interaction of A-nor A.19-dinor,and A-homo-5α-androstane derivatives with the androgen receptor and the epididymal androgen-binding protein

The equilibrium affinity constant for rat prostate androgen receptor and epididymal androgen binding protein (ABP) has been determined for thirty-four potential progestogens. Three A-nor-, four A,19-dinor-, and one A-homo-5α-androstane derivative bind to the androgen receptor (K_D<0.5 μM). Five of these compounds also bind to ABP with an affinity of the same order of magnitude. “Anordrin” (compound $\text{24}$) and “Dinordrins” (compounds $\text{10, 14, 15, 16, 17}$), which are potential female contraceptives, do not bind with high affinity to the androgen receptor or to ABP. The following modifications in A-nor derivatives favour binding to the receptor as compared to ABP: 19-nor substitution (compound $\text{1}$), C-18 methyl homologation (compound $\text{5}$), 2α-ethinylation (compound $\text{22}$). One 2α-allenyl A-nor derivative (compound $\text{25}$) and one A-homo derivative (compound $\text{34}$) bind almost exclusively to ABP. The interaction with either binding protein is decreased by oxidation or esterification of the hydroxyl group at C-17, and by addition of a 17 α-ethinyl group. The latter modifications are likely to increase the specificity of androstane derivatives for receptors other than androgen binding proteins, such as the progesterone receptor.     

Post-castration rebound of an androgen regulated prostatic gene

Summary After castration, the rat dorsolateral prostate M-40 mRNA initially decreased then rebounded to precastrated levels. The cellular site of M-40 expression and its renewed expression after castration was defined by in situ hybridization histochemistry. In situ hybridization with either a ³²P-labeled or biotin-labeled M-40 cDNA probe demonstrated that M-40 mRNA levels were higher in the lateral than dorsal prostate. A second androgen regulated gene, RWB, also was highly expressed in the lateral prostate. The biotinylated cDNA probes provided microscopic resolution of the expressing cells, revealing two distinct morphologies of lateral epithelium which expressed both the M-40 and RWB mRNA. These morphologies appeared in ducts which contained either epithelial cell sheets that were highly convoluted or thinner epithelial cells with a minimal degree of convolution. The RWB mRNA decreased in both cell populations in response to androgen withdrawal. The decline and reappearance of M-40 mRNA also appeared in both epithelial cell types. These data demonstrated that after castration the M-40 mRNA initially decreased as expected for an androgen sensitive gene and then progressed to a fully inducible state. The mechanism of this progression remains to be elucidated.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver

Purified rat liver nuclei were incubated $\text{in vitro}$ with [³H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins.     

Structural polypeptides of primate derived type C RNA tumor viruses

Proteins of gibbon ape lymphosarcoma virus (GaLV) and woolly monkey sarcoma virus, type 1, together with its associated virus ($\text{SSV-1}\text{SSAV-1}$) were analyzed by guanidine-agarose chromatography and the separation patterns were compared with those of mouse and feline type C viruses. GaLV contained five major proteins, including two glycoproteins, whereas lower mammalian viruses contained six major proteins, including two glycoproteins. The molecular weights of the five GaLV proteins closely resembled the molecular weights of the five equivalent lower mammalian viral proteins. $\text{SSV-1}\text{SSAV-1}$ showed a separation pattern similar to GaLV except it contained a low but detectable amount of an additional glycoprotein. Both GaLV and $\text{SSV-1}\text{SSAV-1}$ were deficient in a protein of molecular weight about 15,000 daltons which is found in all known type C viruses of avian, reptilian and lower mammalian species.     

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.     

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

Equilibrium centrifugation of proteins in acidic solutions. Beta-lactoglobulin A in aqueous acetic, propionic, and butyric acids.

Short chain aliphatic acids are almost neutrally buoyant in aqueous solutions, and preferential interaction of macromolecules with these solvent components should not greatly affect apparent molecular weights determined by equilibrium ultracentrifugation. The feasibility of molecular weight estimations using native, neutral pH values of partial specific volume has been tested: equilibrium ultracentrifugation of β-lactoglobulin A (β-LgA) has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. These solutions are highly nonideal because of the extreme Donnan effect. Apparent molecular weights estimated at infinite dilution using the native neutral pH value of the partial specific volume, $\text{v}{}_{\mathrm{p}}$, differed by less than 5% from the monomer formula weight. The 10 m acids appear to be least effective as dissociating agents for β-LgA, with a weak reversible monomer-dimer association suggested in 10 m acetic acid, with significant heterogeneity apparent in 10 m propionic acid, and with a lack of direct solubility in 10 m butyric acid. All the 0.1 m acids and all the 1 m acids were essentially equally effective as dissociating agents, with the exception of 1 m butyric acid which dissolved β-LgA only slowly to give significantly heterogeneous solutions. From these results and from our previous experiments with aldolase (6), it appears feasible to use the native values of $\text{v}{}_{\mathrm{p}}$ to obtain estimates of molecular weights of proteins in aqueous organic acids as dissociating agents.     

Chromatin monomer: Absence of non-histone proteins

Chromatin from a human colon carcinoma cell line has been digested with $\text{Staphylococcal}$ nuclease (E.C.3.1.4.7) and the monomer subunit isolated by sucrose gradient centrifugation. Electrophoresis of the monomer proteins on sodium dodecyl sulfate-polyacrylamide gels revealed an absence of the nuclear non-histone proteins in the isolated monomer.     

Acidic proteins from Saccharomyces cerevisiae ribosomes.

Two dimensional gel electrophoresis of ribosomal proteins from $\text{Saccharomyces}\text{cerevisiae}$ reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH₄Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in $\text{Escherichia}\text{coli}$, rat liver, and $\text{Artemia}\text{salina}$ ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.     

In vitro androgen metabolism in mouse kidney: high 3-keto-reductase (3alpha-hydroxysteroid dehydrogenase) activity relative to 5alpha-reductase

The $\text{in vitro}$ metabolism of testosterone and dihydrotestosterone was studied in slices and cell fractions of mouse kidney. When testosterone was used as substrate, very little metabolism to dihydrotestosterone occurred suggesting very low 5α-reductase activity. When dihydrotestosterone was substrate, it was rapidly converted to 5α-androstane-3α, 17β-diol by a potent 3-keto-reductase. Ninety-five percent of this latter enzyme is located in cytosol and it requires NADPH as cofactor. The 3-keto-reductase may exist in two molecular forms which can be separated by polyacrylamide gel electrophoresis. Form A and B have mean molecular radii which correspond to molecular weights of 38,700 and 28,700, respectively. Sufficient 3-keto-reductase activity is present in cytosol at 0°C to reduce physiological concentrations (2×10^?9 M) of dihydrotestosterone without the addition of cofactor. 3-Keto-reductase activity is higher in intact male than in castrate male or female mice and increases with androgen treatment.From these studies we conclude: (a) The virtual absence of 5α-reductase in mouse kidney is consistent with the thesis that testosterone rather than dihydrotestosterone may be the intracellular androgen in this organ. (b) Kinetic studies which depend upon the $\text{in vitro}$ uptake and retention of dihydrotestosterone by receptor proteins may be difficult to interpret due to rapid metabolism of ligand.     

In vitro androgen metabolism in mouse kidney: High 3-keto-reductase (3α-hydroxysteroid dehydrogenase) activity relative to 5α-reductase

The $\text{in vitro}$ metabolism of testosterone and dihydrotestosterone was studied in slices and cell fractions of mouse kidney. When testosterone was used as substrate, very little metabolism to dihydrotestosterone occurred suggesting very low 5α-reductase activity. When dihydrotestosterone was substrate, it was rapidly converted to 5α-androstane-3α, 17β-diol by a potent 3-keto-reductase. Ninety-five percent of this latter enzyme is located in cytosol and it requires NADPH as cofactor. The 3-keto-reductase may exist in two molecular forms which can be separated by polyacrylamide gel electrophoresis. Form A and B have mean molecular radii which correspond to molecular weights of 38, 700 and 28, 700, respectively. Sufficient 3-keto-reductase activity is present in cytosol at 0°C to reduce physiological concentrations (2×10^?9 M) of dihydrotestosterone without the addition of cofactor. 3-Keto-reductase activity is higher in intact male than in castrate male or female mice and increases with androgen treatment.From these studies we conclude: (a) The virtual absence of 5α-reductase in mouse kidney is consistent with the thesis that testosterone rather than dihydrotestosterone may be the intracellular androgen in this organ. (b) Kinetic studies which depend upon the $\text{in vitro}$ uptake and retention of dihydrotestosterone by receptor proteins may be difficult to interpret due to rapid metabolism of ligand.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.