Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.     

The identification of serum tuftsin by reverse-phase high-performance liquid chromatography and mass spectrometry

We describe for the first time a method for unambiguously identifying the phagocytic stimulating tetrapeptide tuftsin from trypsinized human serum. The method consists of separating serum tuftsin by reverse-phase (RP)-HPLC, collecting HPLC fractions corresponding to the synthetic tuftsin retention time, and then subjecting those fractions to mass spectrometry/mass spectrometry (MS/MS) analysis, which provides optimal molecular specificity to the measurement. Although quantification was not the goal, it was estimated that the amount of tuftsin found by RP-HPLC and MS/MS was in the hundreds of nanograms per milliliter.     

Adenylate cyclase responses to sucrose stimulation in membranes of pig circumvallate taste papillae.

1. Typical adenylate cyclase (AC) responses to guanine nucleotides were found in membranes of pig circumvallate (CV) taste papillae. 2. Sucrose stimulated AC activity in the CV membranes and this stimulation was GTP dependent and tissue specific. 3. The stimulatory effect of sucrose in the CV membranes was dependent on the concentration of membranes used in the AC assay. 4. This study provides the first biochemical data on cellular transduction of taste in the pig, compares positively to preliminary results in cattle and supports recent suggestions for a role of cAMP in sweet taste transduction.     

Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase

Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-Biological Activity Relationships in Alfalfa Antimycotic Saponins: The Relative Activity of Medicagenic Acid and Synthetic Derivatives Thereof Against Plant Pathogenic Fungi1

The antimycotic activity of medicagenic acid and of some synthetic derivatives thereof was tested against plant pathogenic fungi. In general they all possess antimycotic activity. Furthermore, in the case of Sclerotium rolfsii, compounds where the hydroxyl functions of the aglycon remained unchanged (medicagenic acid and its dimethyl ester) or could be enzymically released (3-0-β-D-glucoside of medicagenic acid dimethyl ester) were significantly more active than compounds where these functions were modified by acetylation or methylation. Selective 2-0-methylation of medicagenic acid and comparison of the antimycotic activity of the resulting derivative against S. rolfsii to that of other derivatives suggests that a potential free hydroxyl at position 3 is essential to antimycotic activity.     

Nonreceptor-mediated responses of adenylate cyclase in membranes from liver, muscle, and white and brown adipose tissue of obese (fa/fa) and lean (Fa/) Zucker rats

Adenylate cyclase activity was determined in membranes of liver, muscle, white adipose tissue, and brown adipose tissue (BAT) of lean (Fa/) and obese (fa/fa) Zucker rats. Responses were monitored following beta-adrenergic receptor stimulation and addition of GTP, GTP gamma S, or forskolin. beta-Adrenergic responses in liver, white adipose tissue, and BAT were lower in obese than in lean animals. No such difference was observed in muscle membranes. Production of cAMP after addition of guanine nucleotides was lower in liver and white adipose tissue membranes from obese rats compared with their lean littermates. Synthesis of cAMP in muscle membranes of obese animals after addition of GTP was either not different, or slightly higher, than that observed in muscle membranes from lean animals. Furthermore, production of cAMP after forskolin addition to muscle membranes of obese rats was significantly higher than that observed from lean rats under the same conditions. Interestingly, BAT membranes of obese rats were significantly more sensitive to guanine nucleotide activation than those of lean animals. The results confirm recent findings indicating inferior function of G proteins in liver plasma membranes of obese Zucker rats, and extend this observation to adipose tissue. The present results further suggest that the "nonreceptor" components (e.g., G proteins) responsible for the activation of adenylate cyclase in BAT membranes of obese rats are more responsive to stimulation than those of lean animals. Such sensitivity may be related to and perhaps compensate for the reduced thermogenic activity in the obese Zucker rat during the development of obesity.     

Biocompatible scaffolds based on collagen and oxidized dextran for endothelial cell survival and function in tissue engineering

Angiogenesis is a vital step in tissue regeneration. Hence, the current study aimed to prepare oxidized dextran (Odex)/collagen (Col)-hydrogels with laminin (LMN), as an angiogenic extracellular matrix (ECM) component, for promoting human umbilical vein endothelial cell (HUVEC) proliferation and function. Odex/Col scaffolds were constructed at various concentrations and temperatures. Using oscillatory rheometry, scanning electron microscopy (SEM), and cell viability testing, the scaffolds were characterized, and then HUVEC proliferation and function was compared with or without LMN. The gelation time could be modified by altering the Odex/Col mass ratio as well as the temperature. SEM showed that Odex/Col hydrogels had a more regular three-dimensional (3D) porous structure than the Col hydrogels. Moreover, HUVECs grew faster in the Col scaffold (12 mg/mL), whereas the Odex (30 mg/mL)/Col (6 mg/mL) scaffold exhibited the lowest apoptosis index. Furthermore, the expression level of vascular endothelial growth factor (VEGF) mRNA in the group without LMN was higher than that with LMN, and the Odex (30 mg/mL)/Col (6 mg/mL) scaffold without LMN had the highest VEGF protein secretion, allowing the cells to survive and function effectively. Odex/Col scaffolds, with or without LMN, are proposed as a tissue engineering construct to improve HUVEC survival and function for angiogenesis.     

An Analysis of the Metabolic Theory of the Origin of the Genetic Code

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature. Received: 8 August 1996 / Accepted: 26 December 1996