Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

Summary We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.     

Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.     

Cytosine-rich messenger RNA from carrot root discs

The total poly A⁺ RNA from aerated carrot root discs was further fractionated into a cytosine-rich mRNA fraction by oligo (dG) cellulose chromatography. C-rich mRNA was purified at least 10-fold by this procedure and, when translated in wheat germ lysates, codes for 57 and 53 Kdalton peptides. Translation in double label amino acid mixtures indicates that C-rich mRNA codes for proline-rich peptides which may be precursors to the hydroxyproline-rich glycoprotein synthesized $\text{in}\text{vivo}$ by this tissue.     

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.     

Effects of neuroactive steroids on myelin of peripheral nervous system

Peripheral nervous system (PNS) possess both classical (e.g. progesterone receptor, PR, androgen receptor, AR) and non-classical (e.g. GABA_A receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids. Our data have indicated that neuroactive steroids, like for instance, progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3-diol, stimulate both in vivo and in vitro (Schwann cell cultures), the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). It is important to highlight that the mechanisms by which neuroactive steroids exert their effects on the expression of Po and PMP22 involve different kind of receptors depending on the steroid and on the myelin protein considered. In particular, at least in culture of Schwann cells, the expression of Po seems to be under the control of PR, while that of PMP22 needs the GABA_A receptor.

Because Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the PNS, the present observations might suggest the utilization of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin.     

AZGP1 is androgen responsive and involved in AR-induced prostate cancer cell proliferation and metastasis

Alpha-2-glycoprotein 1, zinc-binding (AZGP1), known as zinc-alpha-2-glycoprotein (ZAG), is a multifunctional secretory glycoprotein and relevant to cancer metastasis. Little is known regarding the underlying mechanisms of AZGP1 in prostate cancer (PCa). In the present study, we report that AZGP1 is an androgen-responsive gene, which is involved in AR-induced PCa cell proliferation and metastasis. In clinical specimens, the expression of AZGP1 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of AZGP1 is upregulated by the androgen-AR axis at both messenger RNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen-responsive elements (AREs) at AZGP1 enhancer; and dual-luciferase reporter assays reveal that the AREs is highly responsive to androgen whereas mutations of the AREs abolish the reporter activity. In addition, AZGP1 promotes G1/S phase transition and cell cycle progress by increasing cyclin D1 levels in PCa cells. Functional studies demonstrate that knocking down endogenous AZGP1 expression in LNCaP and CWR22Rv1 cells largely weaken androgen/AR axis-induced cell migration and invasion. In vivo xenotransplantation tumor experiments also show that AZGP1 involves in androgen/AR axis-mediated PCa cell proliferation. Taken together, our study implicates for the first time that AZGP1 is an AR target gene and is involved in androgen/AR axis-mediated cell proliferation and metastasis in primary PCa.     

Modification of lipophilic proteins in Friend erythroleukemia cells during their differenciation

In mouse erythroleukemia cells (MELC) a lipophilic protein of apparent M. W. 9.5 kdaltons increases during differentiation. This increase is due either to an increase of biosynthesis or to a structural alteration impairing the capacity of the protein to form polymers of apparent high M.W. or favoring its extractability. The increase is related to differentiation and precedes hemoglobin synthesis by at least 1 day. It is not related cells (TFA-11) in which dimethylsulfoxide (DMSO) causes an increase in virus production. As it occurs in cells treated with 4 different inducers, and as the increase is less marked when antagonists of the inducers are also present, it is unlikely that the increase of the 9.5 Kdalton protein is due to an effect of the inducers unrelated to differentiation.     

Elevated level of Wnt5a protein in localized prostate cancer tissue is associated with better outcome

Background

Wnt5a is a non-canonical secreted glycoprotein of the Wnt family that plays an important role in cancer development and progression. Previous studies report that Wnt5a is upregulated in prostate cancer and suggested that Wnt5a affects migration and invasion of prostate tumor cell. This study aimed to evaluate the prognostic value of Wnt5a protein expression in prostate cancer tissue and its potential to predict outcome after radical prostatectomy in patients with localized prostate cancer.

Methodology and Results

Immunohistochemical analysis of a tissue microarray containing prostate specimens of 503 patients with localized prostate cancer showed significantly higher Wnt5a protein expression in cancer compared to benign cores from the same patients (p<0.0001). Patients with high expression of Wnt5a protein had significantly better outcome in terms of time to biochemical recurrence compared to patients with low expression levels (p = 0.001, 95%CI 1.361–3.570, Hazard''s ratio 2.204). A combination of high Wnt5a expression with low levels of Ki-67 or androgen receptor expression had even better outcome compared to all other groups. Furthermore, we found that Wnt5a expression significantly correlated with VEGF and with Ki-67 and androgen receptor expression, although not highly significant. In vitro, we demonstrated that recombinant Wnt5a decreased invasion of 22Rv1 and DU145 cells and that siRNA knockdown of endogenous Wnt5a protein led to increased invasion of 22Rv1 and LNCaP cells.

Conclusion

We demonstrate that preserved overexpression of Wnt5a protein in patients with localized prostate cancer predicts a favorable outcome after surgery. This finding together with our in vitro data demonstrating the ability of Wnt5a to impair the invasive properties of prostate cancer cells, suggests a tumor suppressing effect of Wnt5a in localized prostate cancer. These results indicate that Wnt5a can be used as a predictive marker and that it also is a plausible therapeutic target for treatment of localized prostate cancer.     

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.     

An abundant 56 kD protein with low affinity androgen binding: another member of the steroid/thyroid receptor family?

We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.     

Anabolic responsiveness of skeletal muscles correlates with androgen receptor protein but not mRNA

Anabolic effects of androgens on skeletal muscle are well documented, but the physiological and biochemical bases of these effects are poorly understood. Skeletal muscles that differ in their androgen responsiveness can be used to examine these mechanisms. We compared androgen receptor mRNA and protein levels of the rat levator ani, a perineal skeletal muscle that depends on androgens for its normal maintenance and function with that of the rat extensor digitorum longus, a limb muscle that does not require androgens. Western immunoblotting indicated that androgen receptor protein is significantly elevated in the levator ani relative to the extensor digitorum longus. Surprisingly, steady state androgen receptor mRNA levels were equivalent in these muscles, as determined by Northern blot analysis and quantitative RT-PCR. These results suggest that androgen responsiveness of skeletal muscles is determined by the level of androgen receptor protein in a particular muscle and that androgen receptor protein content is regulated by translational or post-translational mechanisms.     

A 63-kDa protein with androgen-binding activity is not from the androgen receptor.

We have found a new protein in the heart of rat and mice that can be selectively and covalently labelled with the synthetic androgen analog mibolerone. Binding is specific as it can be displaced by excess radioinert ligand. The protein is prominently expressed in liver, kidney, and heart, but not in skeletal muscle. It is water soluble and found in the cytosol. Under denaturing conditions it has a molecular weight of 63,000 and appears on two-dimensional gels with an isoelectric point of 6.3. The protein's affinity for androgen is lower than that of the androgen receptor and it is about 100-fold more abundant than the receptor in the heart. Expression of the protein is not induced by androgen. The presence of this protein in testicular feminization (tfm) mice with a genetical defect of the androgen receptor rules out that it is the androgen receptor or a portion thereof. The biological role of this protein is not yet known.     

A LOW MOLECULAR W eight GLYCOPROTEIN ASSOCIATED WITH ISOLATED MYELIN: DISTINCTION FROM MYELIN PROTEOLIPID PROTEIN

Abstract— A low molecular weight glycoprotein has been demonstrated in myelin isolated from immature rat brains. Both short term and long term fucose incorporation studies have identified this protein in the proteolipid protein region of a sodium dodecyl sulfate, polyacrylamide gel. A 1.7-2.1 fold increase in radioactive fucose in this glycoprotein relative to the major myelin glycoprotein was seen after long term fucose incorporation (21 days) compared to short term incorporation (18–22 h). The demonstration that this fucose-labelled protein is distinguishable from that of proteolipid protein was achieved by a variety of independent techniques. One technique involved a comparison of ether-ethanol extracted, freshly isolated, myelin with myelin extracted with chloroform-methanol. Treatment of isolated myelin with chloroform-methanol results in the solubilization of the proteolopid protein and its subsequent absence on gel electrophoresis while, in contrast, an enhancement of fucose label was observed in the same region of the polyacrylamide gel. Another procedure involved the electrophoretic separation of the radioactive fucose peak from that of proteolipid protein by employing a continuous, phosphate buffered, gel system. Finally carbohydrate analysis by gas-liquid chromatography of a partially purified proteolipid protein fraction did not reveal significant amounts of carbohydrates which are characteristic of glycoproteins. The identification of this minor glycoprotein comigrating with proteolipid protein indicates, therefore, a greater complexity associated with the purified myelin membrane than has been previously demonstrated.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid effects on the gene expression of peripheral myelin proteins

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA(A) receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).     

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Localization of fertility factor SP22 to specific cell types within the anterior pituitary gland

Sperm protein 22 (SP22) was recently identified in the anterior pituitary gland (AP) of male Golden Syrian hamsters using ion trap mass spectrometry. SP22 has been implicated in apoptosis, androgen receptor function, fertility, and ontogeny of early-onset Parkinson's disease. However, the role of SP22 in the pituitary has not been investigated. We cloned the cDNA for full-length SP22 from AP and posterior lobe (posterior pituitary and intermediate lobe) of the pituitary gland in adult male rats and Golden Syrian hamsters, confirming the presence of SP22 mRNA in the AP and posterior lobe. Because gonadal steroids are important regulators of AP function, and SP22 is associated with androgen receptor function, we used Western blots to compare SP22 in the AP of intact and orchidectomized male rats given placebo or a low or high dose of testosterone. SP22 did not differ with treatment, indicating that AP SP22 concentration was not regulated by testosterone. To localize SP22 to specific cells of the AP, mirror-image paraffin sections were labeled against SP22 and either luteinizing hormone (LH)beta, thyroid-stimulating hormone (TSH)beta, prolactin, adrenocorticotropic hormone (ACTH), or growth hormone (GH) using peroxidase-conjugated secondary antibody. Additional sections were colabeled with SP22 and one of the AP hormones using fluorescent secondary antibodies. SP22 colocalized in somatotropes and thyrotropes in rat and hamster. We identified SP22 in a small percentage of corticotropes, gonadotropes, and lactotropes. This is the first report that SP22 mRNA is present specifically in the AP, and SP22 is localized primarily in somatotropes and thyrotropes. SP22 may help regulate AP function and be particularly important for the control of GH and TSH secretion.     

Hormonal regulation of an estramustine-binding protein in the submaxillary gland of the rat

An estramustine binding protein, in many aspects similar to the prostatic secretion protein (PSP), has partly been characterized in the submaxillary gland of the male rat. The [3H]estramustine-macromolecule complex is found in the void volume of a Sephadex G 200 column, indicating a Stokes radius larger than 52 A. The estramustine binding protein is bound to Concanavalin-A, indicating a glycoprotein structure. Like PSP, the macromolecule complex that is bound to Concanavalin-A inhibits the binding of the androgen-receptor complex to DNA-cellulose. The concentration of the protein is decreased following testectomy or estrogen treatment but can be restored to normal values following testosterone administration. These results strongly indicate that the estramustine binding macromolecule in the submaxillary gland belongs to the same group of proteins as PSP. We have earlier proposed a role for PSP as an intracellular regulator of androgen activity. Based on these new results it is tempting to speculate that androgen sensitive glycoproteins may act in the same way in all androgen sensitive tissues.