The aged testis. A good model to find proteins involved in age-related changes of testis by proteomic analysis.

OBJECTIVE: To explore associated proteins involved in age-related changes of the testis and better understand the roles of these proteins in the human testis. STUDY DESIGN: We used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spec trometry analysis to identify differentially expressed proteins between the aged and the normal control groups. The L-lactate dehydrogenase C chain (LDHC) protein, a previous testis-specific protein, was found to be downregulated in the aged testis and was further tested with western blot and immunohistochemical analysis to verify the result of the LDHC protein in 2-DE. RESULTS: Twelve differentially expressed proteins were identified. Among those proteins, 3 were upregulated and 9 were downregulated in the aged group. The results of western blot and immunohistochemical analysis confirmed the expression of LDHC downregulation in the aged testis. Some proteins identified had little well-known function in the human testis, as follows: AKR7A3, FDXR, PGAM1, SEPT2 and HMGCS2. CONCLUSION: Our results imply that the aged testis can be a good model to find associated proteins involved in age-related changes of the testis. It can be useful to understand the roles of those proteins in the testis.

[The highly expressed secreted phosphoprotein 1 gene in prostate cancer metastasis: a microarray-based bioinformatic analysis].

OBJECTIVE: To investigate the composition, function, and regulatory mechanisms of the secreted phosphoprotein 1 (SPP1) gene in metastatic prostate cancer. METHODS: We obtained the data about the whole genomic expression profiles on prostate cancer metastasis from the GEO database, and performed data-mining and bioinformatic analysis using BRB-Array Tools and such softwares as Protparam, MotifScan, SignalP 4.0, TMHMM, NetPhos2.0, PredictProtein, GO, KEGG, and STRING. RESULTS: Totally, 73 co-expressed differential genes in prostate cancer metastasis were identified, 21 up-regulated and 52 down-regulated (P <0.01). Bioinformatic analysis indicated that the highly expressed SPP1 gene encoded 314 amino acids and contained 2 N-glycosylation sites, 8 casein kinase II phosphorylation sites and 3 protein kinase C phosphorylation sites, playing essential roles in extracellular matrix (ECM) binding, ossification, osteoblast differentiation, cell adhesion, PI3K-Akt signaling pathway, focal adhesion, Toll-like receptor signaling pathway, and ECM-receptor interaction. CONCLUSION: The bioinformatic method showed a high efficiency in analyzing microarray data and revealing internal biological information. SPP1 may play an important role in prostate cancer metastasis and become a novel biomarker for the diagnosis of prostate cancer metastasis and a new target for its treatment.

[Correlation of aging with psychological and organic ED: nocturnal electrobioimpedance volumetric assessment of 83 cases].

OBJECTIVE: The ratio of psychological to organic ED changes with aging. This study aimed to analyze the results of nocturnal electrobioimpedance volumetric assessment (NEVA) for ED patients of different age groups and their significance in the diagnosis of ED. METHODS: A total of 83 ED patients were divided into 4 age groups (< or = 29 yr, 30 -39 yr, 40 -49 yr and > or = 50 yr) and detected for nocturnal penile tumescence (NPT) by NEVA. RESULTS: Thirty-four of the cases were diagnosed as organic ED, and the other 49 as psychological ED. With the increase of age, the former was increased from 30.3% in the < or = 29 yr group to 60.0% in the > or = 50 yr group, while the latter decreased from 69.7% to 40.0%. CONCLUSION: The percentage of organic ED tends to grow with the increase of age, while that of psychological ED is just the opposite.

[Single- and two-layer gradient centrifugation in sperm separation: comparison and appraisal].

OBJECTIVE: To appraise the effect of single- and two-layer Percoll density gradient centrifugation in sperm separation. METHODS: Twenty semen specimens underwent single-(50%) and two-layer (90% and 45%) density gradient centrifugation, respectively. The sperm class analyzer (SCA) was used to analyze sperm density, motility and dynamic parameters and round cell density before and after the treatment. RESULTS: After separation, the sperm recovery rate of the single-layer method was (65.5 +/- 12.8)%, significantly higher than that of the two-layer method (P < 0.01). The percentages of grade a sperm of the single- and two-layer method were significantly higher than pre-treatment (P < 0.05, P < 0.01), that of the single-layer was significantly lower than that of the two-layer method (P < 0.05), but the percentage of grade c sperm of the former was significantly higher than that of the latter (P < 0.05). Compared with pre-treatment, the percentage of grade a + b sperm of the two-layer method was significantly higher (P < 0.05), while that of the single-layer method showed no significant difference (P > 0.05), and the round cell density of both the methods was significantly lower (P < 0.05, P < 0.01), with no significant differences between the two methods (P > 0.05). CONCLUSION: The single-layer method yields a higher rate of sperm recovery and causes little change in the sperm motility, while the two-layer method effects a lower rate and significantly improves sperm motility. Both the methods can efficiently separate sperm from round cells, and each has its own advantages and its application value in in vitro treatment of sperm.

Phosphoribosyl pyrophosphate synthetases 2 knockdown inhibits prostate cancer progression by suppressing cell cycle and inducing cell apoptosis.

Phosphoribosyl pyrophosphate synthetases 2 (PRPS2) protein function as nucleotide synthesis enzyme that plays vital roles in cancer biology. However, the expression profile and function of PRPS2 in prostate cancer (PCa) remain to be identified. Here we investigated the expression of PRPS2 protein in human PCa and paired normal tissues by immunohistochemistry, meanwhile the regulatory effects on cell proliferation, apoptosis and growth of xenograft tumors in nude mice were evaluated in PCa cells with PRPS2 depletion. Moreover, the signaling pathways were also explored by western blot analysis and quantitative polymerase chain reaction assays. We found that PRPS2 was dramatically upregulated in prostate adenocarcinoma tissues in comparison with normal tissues, and that increased PRPS2 was linked intimately to advanced clinical stage and pT status. Functional experiments showed that knockdown of PRPS2 significantly suppressed cell growth both in vitro and in vivo. In addition, depletion of PRPS2 induced G1 phase cell cycle arrest and elevated cell apoptosis. Silencing of PRPS2 resulted in the decreased expression of Bcl­2 and cyclinD1 and increased levels of Bax, cleavage of caspases­3, caspases­9 and PARP. Furthermore, we also detected PRPS2 expression was significantly induced after DHT treatment, which implied the important role of PRPS2 in oncogenesis of PCa. Taken together, our findings elucidated that PRPS2 may be a potential novel candidate for PCa therapy.

[Minute dose of tadalafil for nocturnal penile tumescence].

OBJECTIVE: To investigate the efficacy of tadalafil on nocturnal penile tumescence (NPT). METHODS: Thirty-four patients with organic erectile dysfunction (ED) were treated with oral tadalafil at the dose of 10 mg/3 d before bedtime. A month later, 14 of the patients were observed for NPT by nocturnal electrobioimpedance volumetric assessment (NEVA). RESULTS: The parameters of erectile function significantly improved in the 14 patients (P < 0.05). CONCLUSION: Oral administration of minute dose of tadalafil can improve NPT in organic ED patients.

[Literature-mining and bioinformatic analysis of androgen-independent prostate cancer-specific genes].

OBJECTIVE: To compare the differences of the gene expressions in androgen-independent and androgen-dependent prostate cancer (ADPC), gain a deeper insight into the molecular mechanism of androgen-independent prostate cancer (AIPC), and find effective means for its clinical diagnosis and treatment. METHODS: Eats of genes highly-associated with prostate cancer were obtained by mining PubMed with the FACTA tool, and the specifically expressed genes in AIPC were analyzed with a set of bioinformatic tools including GATHER, PANTHER, STRING and ToppGene. RESULTS: A total of 128 genes specifically expressed in AIPC were identified, as compared with 23 that were specific to ADPC. Bioinformatic analysis showed the essential roles of AIPC-specific genes in such important biological processes as cell signal transduction, cell adhesion, apoptosis, oncogenesis, cell proliferation and cell differentiation. CONCLUSION: Such genes as MMPJ, EGFR, MMP2, ADM, MIF, IGFBP3, 112, MET, BAD, RHOA, SPP1, EP300, SMAD3, RAE1, PTK2, and TGFB2 may play important roles in transforming ADPC into AIPC.

[Mutation of MTCYB and MTATP6 is associated with asthenospermia].

OBJECTIVE: To explore the correlation of the mutation of MTCYB and MTATP6 genes in sperm mitochondria with asthenospermia. METHODS: We extracted mtDNA from 80 semen samples of asthenospermia and 20 of normal sperm motility, amplified the MTCYB and MTATP6 genes by PCR, and analyzed their mutation by sequencing and BLAST matching. RESULTS: The deletion of both MTCYB and MTATP6 were detected in 20 of the 80 asthenospermia samples, MTCYB deletion in 16 and MTATP6 deletion in 4, accounting for 20% and 5% respectively. Sequencing and BLAST matching revealed G8887A mutation in the MTATP6 gene in the asthenospermia samples, with a mutation rate of 20%, while no regular mutation was noted in MTCYB. Neither significant deletion nor mutation was observed in any of the 20 samples of normal sperm motility. CONCLUSION: Both the deletion and mutation of MTCYB and MTATP6 genes in sperm mitochondria might affect sperm motility in adults.

Periostin is essential for periodontal ligament remodeling during orthodontic treatment.

Orthodontic tooth movement is a process stimulated and maintained by external tensile stress; periodontal ligament remodeling serves an important role during this process. However, the function and underlying mechanism of periostin (PN) during orthodontic periodontal ligament remodeling remain unclear. The present study established in vitro and in vivo models of orthodontic treatment to investigate the expression levels of PN under conditions of external tensile stress load. These results indicated that tensile stress load increased the expression levels of PN in mouse peridontal ligaments and human periodontal ligament cells (hPDLCs), during orthodontic tooth movement. Furthermore, the present study demonstrated that the expression levels of PN were regulated by transforming grown factor ß, and that PN promotes type I collagen and α­smooth muscle actin expression levels in hPDLCs. Therefore, PN may be essential for periodontal ligament remodeling during orthodontic treatment, and therefore may represent a potential therapeutic target.

[Research of the spermatozoal gene expression with gene microarrays].

OBJECTIVE: To perform the detection of spermatozoal gene expression in order to accelerate the study of spermatozoal molecular biology. METHODS: To collect the healthy adults sperm and lymphocytes respectively, and then to extract the total RNAs from them by RNeasy mini kit (QIAGEN) or Trizol reagent. Corresponding cDNAs were produced, digested, ligated, finally labeled with Cy3 (sperm) and CyS (lymphocyte) in the course of RD amplifying reactions. Hybridization with self-made microarrays contained 560 probes was carried out after the labeled cDNAs pured by PCR Product Purification Kit. RESULTS: Among the 560 probes, 72 genes were up-regulated, 321 genes were down-regulated, the others had no different expression. Furthermore, genes associated with replication, transcription, translation and regulative functions were non-different expression or down-regulated, and those belonged to the spermatogenesis associated, sperm associated antigen were up-regulated, but those involved in the glycolysis were up-regulated, in the oxidative phosphorylation were down-regulated. CONCLUSION: It had successfully confirmed that there were a plenty of genes expressed in sperm, furthermore the genes expressed were accorded to spermatozoal functions and characteristics.

Clinical effect of removable partial denture prepared by Saddle-Lock hidden-clasp.

OBJECTIVE: To study the method for preparing removable partial denture using Saddle-Lock hidden-clasp and evaluate its clinical effect. METHOD: Casting process was adopted for the preparation of the clasp, which was fixed at the points on the mesial and distal surfaces of the abutment tooth. The clinical records of 305 patients treated with such removable partial dentures were reviewed. RESULTS: Within a follow-up period of 6 years, 295 of the 305 patients retained good abutment condition and were satisfied with the denture that was capable of easy wearing and removing. CONCLUSIONS: Removable partial denture made by Saddle-lock hidden-clasp exhibits good performance in fixation and stability. As an innovation of traditional design of partial denture clasp, it is simple to prepare and well received by the patients with good clinical effect.

[An initial examination of the spermatozoal gene expression profile].

OBJECTIVE: To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles. METHODS: The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends. According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli. The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides. The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples. RESULTS: Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray. CONCLUSIONS: Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality.

[Mining the specifically expressed genes in sperms based on the bioinformatics methods].

OBJECTIVE: To analyze the specifically expressed genes in sperms for better understanding of the molecular characteristics of sperms. METHODS: The hybridization data the genes in the sperms, oocytes and 10 normal tissues were retrieved from the GEO database to identify the genes expressed specifically in sperms and the patterns of their regulation using such bioinformatic tools as GATHER, PANTHER and DAVID. RESULTS AND CONCLUSIONS: Comparison of the spermatozoal gene expression profiles with those of the normal tissues identified 8998 differentially expressed probes, among which 25 genes were up-regulated by over 200 folds in the sperms. Comparison of the gene expression profiles between the oocytes and normal tissues resulted in the identification of 8981 differentially expressed probes. Of the 1709 up-regulated genes in the sperm with a ratio>5, 1218 genes showed similar expressions in the oocytes and the normal tissues, and 129 were up-regulated and 362 down-regulated in the oocytes. The 362 genes up-regulated in the sperms but down-regulated in the oocytes were involved mainly in protein modification and metabolism and nucleic acid metabolism, but very few participated in the intracellular signaling pathways. Numerous transcriptional factors containing the KRAB domain and receptor- independent serine/threonine kinase were specifically overexpressed in sperms, and the a very high proportion of the genes specifically overexpressed in the sperms coincided with the overexpressed genes in the neural stem cells and embryonic stem cells. The genes involved in the glycolysis were down-regulated in the sperms. These findings in the genes specifically expressed in the sperms by data mining using bioinformatic methods may provide better insight into the molecular characteristics of the sperms.

[Identification of human testicular embryonal carcinoma proteins by two-dimensional gel electrophoresis and mass spectrometry].

OBJECTIVE: To separate and identify human testicular embryonal carcinoma proteomics using two-dimensional electrophoresis (2-DE) and mass spectrometry. METHODS: Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis was used to separate the total proteins of the samples. After silver staining, PDQuest 7.30 image analysis software was applied to analyze the 2-DE images. Three of the proteins highly expressed in human testicular embryonal carcinoma were identified by matrix-assisted laser adsorption/ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS). RESULTS: 2-DE effectively screened the differentially expressed proteins in the carcinoma tissues. Three proteins highly expressed in the carcinoma were successfully identified. CONCLUSION: The proteins of human testicular embryonal carcinoma can be effectively separated and analyzed using 2-DE and mass spectrometry. Proteomic analysis offers a new means for further study of this carcinoma.

[Expression of MMP1 mRNA in oral squamous cell carcinoma and paired normal tissues].

OBJECTIVE: To investigate the mRNA expression of matrix metalloproteinase 1 (MMP1) gene in oral squamous cell carcinoma (OSCC) and the paired normal tissues. METHODS: The differential expression of MMP1 mRNA between 30 OSCC and paired normal tissues were detected with reverse transcription-PCR (RT-PCR). RESULTS: The relative expression level of MMP1 mRNA in the OSCC tissues showed a 3.26-fold increase in comparison with that in the paired normal tissues (4.06-/+0.52 vs 1.24-/+0.17, P<0.0001). In the 30 OSCC tissues, the relative expression level of MMP1 mRNA was higher in histological grade II/III tissues (4.31-/+0.68) than in grade I (3.87-/+0.57) tissues, higher in OSCC in advanced stages (III/IV) than in tumors in early stages (I/II) (4.18-/+0.67 vs 3.65-/+0.53), and also higher in OSCC with cervical lymph node invasion than in those without cervical lymph node invasion (4.32-/+0.71 vs 3.91-/+0.51), but these differences were not statistically significant (P>0.05). CONCLUSION: MMP1 gene may play a role in local invasion of OSCC, and can serve as a potential biomarker molecule for diagnosis, treatment and prognostic evaluation of OSCC, with also clinical value for OSCC classification.