iSP-RAAC: Identify Secretory Proteins of Malaria Parasite Using Reduced Amino Acid Composition.

BACKGROUND: As the pathogen of malaria, malaria parasite secretes a variety of proteins for its growth and reproduction. OBJECTIVE: The identification of the secretory proteins of malaria parasite has crucial reference significance for the development of anti-malaria vaccines as well as medicine. METHODS: In this study, a computational classification method was developed to identify the secreted proteins of Plasmodium. Amino acid composition, dipeptide composition, and tripeptide composition as well as reduced amino acids alphabets were proposed to illuminate protein sequences. We further used SVM to train and predict respectively and optimized the features. RESULTS: 74 types of reduced amino acids alphabets were employed to predict secretory proteins. The results showed that the accuracy improved to 91.67% with 0.84 Mathew's correlation coefficient (MCC) by dipeptide composition, and the highest prediction accuracy reached 92.26% after feature selection, which demonstrated that our method is prominent and reliable in the field of malaria parasite secreted proteins prediction. CONCLUSION: A intuitive web server iSP-RAAC (http://bioinfor.imu.edu.cn/isppseraac) was established for the convenience of most experimental scientists.

Development and validation of analytical methodologies for the quantification of PCK3145 and PEG-PCK3145 in mice.

PCK3145 is an anti-metastatic synthetic peptide against prostate cancer. The objective of the study is to develop and validate novel and sensitive methods for the determination of PCK3145 and Pegylated PCK3145 in mouse plasma. An LC-MS/MS method was developed and validated for the determination of PCK3145 giving high sensitivity and linearity in the range of 0.125-4.0 µg/mL. PCK3145 characterised by short half-life, therefore, it was conjugated with the poly ethylene glycol (PEG). However, LC-MS/MS has been more difficult to apply for the quantitative analysis of PEGylated peptides due to the large size. A UHPLC-UV method was developed and validated for the determination of PEG-PCK3145, with linearity of 0.05-2.0 mg/mL. In order to further improve the sensitivity for the detection of PEG-PCK3145, an indirect ELISA method was used. It was found that this method was capable of detecting PCK3145 through the quantification of PEG with excellent sensitivity found at 0.132 ng/mL. The in vitro proteolytic stability of PCK3145 and PEG-PCK3145 in mouse plasma and whole blood was studied by LC-MS/MS and UHPLC, respectively. The LC-MS/MS and ELISA methods can be applied for monitoring levels of PCK3145 in mouse plasma for in vivo pharmacokinetic and bioavailability animal studies.

Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer.

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.

Cysteine-rich secretory protein 3 and ß-microseminoprotein on prostate cancer needle biopsies do not have predictive value for subsequent prostatectomy outcome.

OBJECTIVES: • To investigate whether cysteine-rich secretory protein 3 (CRISP-3) and/or ß-microseminoprotein (ß-MSP) expression in diagnostic prostate needle biopsies have predictive value for prostate cancer (PC) on radical prostatecomy (RP). • To evaluate their potential clinical implementation in a preoperative setting. PATIENTS AND METHODS: • In total, 174 participants from the European Randomized Study of Screening for Prostate Cancer, Rotterdam section, treated by RP for PC were included in the present study. • CRISP-3 and ß-MSP immunohistochemistry was performed on corresponding diagnostic needle biopsies. • Outcome was correlated with clinicopathological parameters (prostate-specific-antigen, PSA; number of positive biopsies; Gleason score, GS; pT-stage; surgical margins at RP) and significant PC at RP (pT3/4, or GS > 6, or tumour volume ≥ 0.5 mL) in the total cohort (n= 174) and in a subgroup with low-risk features at biopsy (PSA ≤ 10 ng/ml, cT ≤ 2, PSA density <0.20 ng/mL/g, GS < 7 and ≤ 2 positive biopsy cores; n= 87). RESULTS: • ß-MSP and CRISP-3 expression in PC tissue was heterogeneous, with variable staining intensities occurring in the same tissue specimen. • High expression of ß-MSP significantly correlated with GS < 7 at RP; it was not a predictor for significant PC at RP neither in the total group (n= 174; odds ratio, OR, 0.319; 95% confidence interval, CI, 0.060-1.695; P= 0.180), nor in the low-risk group (n= 87; OR, 0.227; 95% CI, 0.040-1.274; P= 0.092). • CRISP-3 expression was not related to clinicopathological parameters, and did not predict significant PC at RP in the total group (n= 174; OR, 1.056; 95% CI, 0.438-2.545; P= 0.904) or the low-risk group (n= 87; OR, 1.856; 95% CI, 0.626-5.506; P= 0.265). CONCLUSIONS: • High ß-MSP expression correlated with low GS in subsequent RP specimens, supporting the view that ß-MSP exerts a tumour-suppressive effect. • No significant prognostic value of ß-MSP or CRISP-3 in prostate needle biopsies for significant PC at RP was found. • ß-MSP or CRISP-3 do not have additional value in the therapeutic stratification of patients with PC.

Polymorphisms at the Microseminoprotein-beta locus associated with physiologic variation in beta-microseminoprotein and prostate-specific antigen levels.

BACKGROUND: rs10993994, a single nucleotide polymorphism (SNP) at the genetic locus encoding beta-microseminoprotein (beta-MSP), is associated with both prostate cancer risk and levels of blood prostate-specific antigen (PSA), a biomarker used in prostate cancer screening. Therefore, we wished to determine the association between SNPs at MSMB, the gene encoding beta-MSP, and the levels of prostate-produced biomarkers beta-MSP, PSA, and human kallikrein 2 (hK2) in blood and semen. METHODS: Blood and semen from 304 healthy young Swedish men (ages 18-21) were assayed for beta-MSP, PSA, and hK2. SNPs around MSMB were genotyped from matched DNA and analyzed for quantitative association with biomarker levels. Empirical P values were multiple test-corrected and the independence of each SNP's effect was determined. RESULTS: rs10993994 was significantly associated with the blood and semen levels of beta-MSP (both P < 1.0 x 10(-7)) and PSA (P = 0.00014 and P = 0.0019), and semen levels of hK2 (P = 0.00027). Additional copies of the prostate cancer risk allele resulted in lower beta-MSP but higher PSA levels, and singly explained 23% and 5% of the variation seen in semen beta-MSP and PSA, respectively. Additional SNPs at MSMB are associated with beta-MSP and PSA independently of rs10993994. CONCLUSIONS: SNPs at MSMB correlate with physiologic variation in beta-MSP and PSA levels in the blood and semen of healthy young Swedish men. In particular, rs10993994 has a strong effect on beta-MSP levels. IMPACT: Our results suggest a mechanism by which rs10993994 might predispose to prostate cancer and raise the possibility that genetic variation might need to be considered in interpreting the levels of these biomarkers.

In-depth proteomic analyses of direct expressed prostatic secretions.

It is expected that clinically obtainable fluids that are proximal to organs contain a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue and thus represent potential sources for biomarker discovery, investigation of tissue-specific biology, and assay development. The prostate gland secretes many proteins into a prostatic fluid that combines with seminal vesicle fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed prostatic secretion (EPS) fluids. In the current study, MudPIT-based proteomics was applied to EPS obtained from nine men with prostate cancer and resulted in the confident identification of 916 unique proteins. Systematic bioinformatics analyses using publicly available microarray data of 21 human tissues (Human Gene Atlas), the Human Protein Atlas database, and other published proteomics data of shed/secreted proteins were performed to systematically analyze this comprehensive proteome. Therefore, we believe this data will be a valuable resource for the research community to study prostate biology and potentially assist in the identification of novel prostate cancer biomarkers. To further streamline this process, the entire data set was deposited to the Tranche repository for use by other researchers.

Identification of rat prostatic secreted proteins using mass spectrometric analysis and androgen-dependent mRNA expression.

Rats have been used to study the function and development of the mammalian prostate. Identification of prostatic secreted proteins is important in order to better understand their physiological function. Previous investigations have showed that prostatein, cysteine-related protein 1, and kallikrein S3 are in the ventral prostate (VP), whereas the proteins probasin, prostate secretory peptide 94, transglutaminase 4, and carbonic anhydrase II are produced in the lateral prostate, dorsal prostate (DP), and anterior prostate. They are also useful markers when looking at androgen dependency as well as prostate-specific expression. Although some of the rat prostatic proteins have been investigated well, the overall protein expression profile of the prostate has not been examined. In the present study, the secretions from the rat prostate were subjected to 2-dimensional gel electrophoresis followed by mass spectrometric analysis. In addition to the previously known proteins, proteome analysis revealed several new secreted proteins, including spermine-binding protein and a protein similar to immunoglobulin-binding protein. In addition, epididymal secreted protein 1 and peroxiredoxin 6 were found in the DP, while glucose-regulated protein 78 was identified in all lobes of the prostate. Castration of the animals led to a decrease in the mRNAs of all of these secreted proteins. While the mRNAs of prostatic proteins became almost completely absent in the VP, the reductions in the other lobes were limited. A novel view of rat prostate secretion from our results should contribute to an understanding of the biological functions of the prostate gland.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Identification of prostatic-secreted proteins in mice by mass spectrometric analysis and evaluation of lobe-specific and androgen-dependent mRNA expression.

Rats and guinea pigs have frequently been used to study the development of the prostate and the mechanism of androgen action, but the mouse prostate has also become an attractive model for prostate research, because an enormous range of genetically altered mice is now available. However, the secretion of proteins in the mouse prostate has not yet been thoroughly investigated. In the present study, major secreted proteins from the ventral prostate (VP), dorso-lateral prostate (DLP), and anterior prostate (AP) of mice were identified by means of 2D-gel electrophoresis followed by MALDI-TOF mass spectrometric analysis. A quantitative reverse transcriptase-PCR method was further employed to examine the androgen-dependent transcriptional regulation of the identified proteins. Proteome analysis revealed that the VP secretes spermine-binding protein, serine protease inhibitor Kazal type-3, and a 91 kDa hypothetical scavenger receptor (AK035662). DLP and AP secrete a protein similar to immunoglobulin-binding protein, immunoglobulin-binding protein-like protein, and one of the experimental autoimmune prostatitis antigen proteins (EAPA2). Peroxiredoxin-6, glucose-regulated protein 78, zinc-alpha2-glycoprotein, and phospholipase Calpha are also secreted. Castration of animals led to a decrease in the mRNAs of these secreted proteins, although the extents of changes varied greatlyamong different lobes. We present here an outlined view of mouse prostate secretion, which should contribute to an understanding of the biological functions of the prostate gland, as well as the androgen dependency of prostate secretion.

Increased intratumoral expression of prostate secretory protein of 94 amino acids predicts for worse disease recurrence and progression after radical prostatectomy in patients with prostate cancer.

OBJECTIVES: To examine the impact of prostate secretory protein of 94 amino acids (PSP94) expression within the primary tumor on disease recurrence and progression using radical prostatectomy specimens from patients with adenocarcinoma of the prostate. METHODS: PSP94 immunohistochemistry was performed on 59 radical prostatectomy specimens. The degree of PSP94 expression was reported as either present (group 1) or absent (group 2). Clinical data, including survival outcome measures, were correlated with PSP94 expression. RESULTS: The time to disease progression for group 1 was shorter compared with that for group 2 (P = 0.042). Disease-free survival was also less in group 1 than in group 2 (P = 0.033). Multivariate analyses demonstrated that PSP94 expression was an independent prognostic factor for a shorter interval to disease progression (P = 0.046) and disease-free survival (P = 0.049). CONCLUSIONS: PSP94 expression in radical prostatectomy tumor specimens appears to be associated with worsened survival outcomes and may provide additional prognostic information.

Anemia in patients on combined androgen block therapy for prostate cancer.

AIM: To study the effect of combined androgen block therapy on hemoglobin and hematocrit values in patients with prostate cancer. METHODS: One hundred and thirty-six patients with adenocarcinoma of prostate were treated with combined androgen block (orchiectomy and flutamide 250 mg, tid). Complete blood counts were determined before and after 1, 2, 3, 6, 9 and 12 months of therapy. RESULTS: The hemoglobin and hematocrit levels declined significantly in all patients and at all the time points after treatment (P<0.05). CONCLUSION: Prostate cancer patients treated with combined androgen block would develop obvious anemia. Recombinant human erythropoietin can be used to treat patients with severe anemia.

Expression study of three secretory proteins (prostatic secretory protein of 94 amino acids, probasin, and seminal vesicle secretion II) in dysplastic and neoplastic rat prostates.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94), probasin, and seminal vesicle secretion II (SVSII) are the three major proteins secreted by the lateral lobe of the rat prostate gland. Among these proteins, rodent PSP94 but not probasin and SVSII has a human homologue and it is also a major secretory protein of the human prostate, in addition to prostatic acid phosphatase and prostate-specific antigen. METHODS: In this study, we examined and compared the mRNA expression of these three secretory markers in three rat models of prostate cancer including the sex steroid-induced dysplasia (prostatic intraepithelial neoplasia or PIN) in Noble (Nb) rat model, an androgen-independent Nb rat prostatic tumor (AIT) and Dunning rat prostatic adenocarcinomas (both androgen-dependent and -independent) by in situ hybridization (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemistry. RESULTS: The transcripts for the three markers were highly expressed in the secretory epithelium of normal lateral prostate (LP). Their hybridization signals became reduced in the epithelial cells in the low-grade PINs and significantly weakened or lost in the high-grade PINs induced in the LP. Interestingly, we observed that some dysplastic cells located at the basal compartment of the PIN lesions, and nests of outpouching epithelial cells in the vicinity of PINs, expressed positive hybridization signals of three markers. In the adenocarcinoma, signals of probasin but not PSP94 and SVSII were detected. No hybridization signals were detected in both Dunning and AIT tumors. By RT-PCR, transcripts for these proteins were still detected but significantly reduced in the Dunning tumors, whereas in the AIT tumor, only SVSII transcripts were detected. Immunohistochemistry of PSP94 also showed a reduced staining in the PIN lesions, but no immunoreactivity was seen in the rat prostatic tumors. CONCLUSIONS: The mRNA expression of the three prostatic secretory markers were decreased in the hormone-induced PINs and in two rat prostatic tumors, indicating that the androgen-regulated secretory differentiation was impaired during the development of the premalignant lesion and further reduced in advanced tumors. The abnormal expression pattern of these secretory markers and androgen receptor (AR) in the basal compartment of the PIN lesions suggests that there is a population of cell types with secretory phenotype appearing in the basal cell layer during the early malignant transformation of the prostatic epithelium.