Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (～1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.     

Crystal structure of human cytochrome P450 2D6 with prinomastat bound

Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 ? resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F', G", G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.     

A combination of intradermal jet-injection and electroporation overcomes in vivodose restriction of DNA vaccines

Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.     

An immunohistochemical approach to monitor the prolactin-induced activation of the JAK2/STAT5 pathway in pancreatic islets of Langerhans.

This study examined whether an immunohistochemical method examining the subcellular localization of STAT5 could be used to characterize the activation of the JAK2/STAT5 pathway by prolactin (PRL) in intact cells or tissues. In the Ins-1 beta-cell line, STAT5A and STAT5B were distributed almost equally in the cytoplasm and the nucleus in unstimulated cells. STAT5A was also detected along the border of cells and in the perinuclear region. After exposure to PRL, the redistribution from the cytoplasm to the nucleus was much higher for STAT5B compared to STAT5A. This translocation represented 12% of the STAT5A and 22% of the STAT5B originally located in the cytoplasm before stimulation. In isolated rat islets of Langerhans, PRL stimulated the nuclear translocation of both STAT5A and STAT5B only in beta-cells. The expression of the PRL receptor only by beta-cells was confirmed with a rabbit polyclonal antiserum raised against the rat PRL receptor. It was estimated that 4% of STAT5A and 9% of STAT5B originally located in the cytoplasm was translocated to the nucleus after stimulation. The presence of a functional JAK2/STAT5 signaling pathway in all islet cells was demonstrated by the nuclear translocation of STAT5B in all islet cells (i.e., alpha-, beta-, and delta-cells) after stimulation with fetal calf serum. The nuclear translocation and tyrosine phosphorylation of STAT5B was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 hr, and prolonged activation after 4 hr. In contrast, the tyrosine phosphorylation of STAT5A was also biphasic but its nuclear translocation peaked within 30 min and was then reduced to a level slightly above that observed before PRL stimulation. This method is able to detect changes in STAT5 activation as small as 2% of the total cell content. These observations demonstrate the utility of this approach for studying the activation of STAT5 in a mixed population of cells within tissues or organs. In addition, the dose response for the nuclear translocation of STAT5B in normal beta-cells was similar to those for changes in proliferation and insulin secretion in isolated rat islets. Therefore, the subcellular localization can be used to monitor the activation of STAT5 and it may be a key event in the upregulation of the pancreatic islets of Langerhans during pregnancy.     

Regulation of idiotope expression. I. The effect of antigen dose on expression of certain T15 idiotopes during primary IgM response to S. pneumoniae R36a

Clonal heterogeneity among B cells reactive to the same epitope may be determined through differences in idiotypy. It appears that clones bearing distinct idiotopes may constitute functionally distinct subpopulations. Data suggest that idiotopically distinct clones of PC-reactive B cells may be regulated independently of one another. We have looked to see whether individual T15+ clones may also differ in their requirements for activation. Here we examine the effect of immunizing doses of antigen on expression of two T15 idiotopes, B36-82 and B39-38, during both in vivo and in vitro primary responses to Streptococcus pneumoniae R36a (Pn) in CB-20 mouse strain. The idiotopes were detected on the specific antibody plaque-forming cells (PFC) by using monoclonal anti-idiotopic antibodies. We find that distinct patterns of idiotope expression are generated by stimulation with different doses of antigen. Immunization with suboptimal and super-optimal doses of Pn produced responses dominated by PFC expressing both idiotopes, whereas PFC induced by optimal antigen concentrations were primarily B36-82+ and B39-38-. These data indicate that the varying of antigen concentration may induce the response of different B cells bearing distinct idiotypes.     

Inverse relationship between Leydig cell density and metastatic potential of prostatic adenocarcinoma.

PURPOSE: Evaluate the relationship between metastatic potential of prostatic adenocarcinoma (PC) and testicular Leydig cell density. MATERIALS AND METHODS: Tissue samples from 111 men, age 52-85, with PC and bilateral orchiectomy were evaluated for Leydig cell density. The patients were divided into two groups: Group A were patients with metastasis (n = 36) and Group B were patients without metastasis (n = 75). Leydig cell density was determined by direct manual microscopic cell count on the tissue sections. The means of cell counts by four pathologists, expressed as cell/0.78 mm2 were used for analysis. The normally distributed data were analyzed by two-tail Student's t-test. Thirty-eight age-compatible autopsy cases who died of unrelated causes served as normal controls. RESULTS: The mean of Leydig cell count in group A patients was 14.43 (14.43+/-1.19 SE). Mean of Group B was 47.05 (47.05+/-4.05 SE) whereas normal controls displayed a mean of 48.66 (48.66+/-2.94 SE). Group A was significantly different from control (p < 0.00001). Group A and Group B were also significant different (p < 0.001) whereas control was not significantly different from Group B (p > 0.75). CONCLUSIONS: Patients with metastatic adenocarcinoma of prostate, as a group, have a significantly lower Leydig cell density than patients without metastasis or patients without PC in compatible age groups. The hormonal relationship between this observation is however unknown. One possible explanation is that PC subpopulation with metastatic potential may require different level of endogenous androgen or are androgen-independent.     

Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.