Hesperidin modulates the rhythmic proteomic profiling in Drosophila melanogaster under oxidative stress.

The circadian clock regulates vital aspects of physiology including protein synthesis and oxidative stress response. In this investigation, we performed a proteome-wide scrutiny of rhythmic protein accrual in Drosophila melanogaster on exposure to rotenone, rotenone + hesperidin and hesperidin in D. melanogaster. Total protein from fly samples collected at 6 h intervals over the 24 h period was subjected to two-dimensional gel electrophoresis and mass spectrometry. Bioinformatics tool, Protein ANalysis THrough Evolutionary Relationships classification system was used to the determine the biological processes of the proteins of altered abundance. Conspicuous variations in the proteome (151 proteins) of the flies exposed to oxidative stress (by rotenone treatment) and after alleviating oxidative stress (by hesperidin treatment) were observed during the 24 h cycle. Significantly altered levels of abundance of a wide variety of proteins under oxidative stress (rotenone treatment) and under alleviation of oxidative stress (rotenone + hesperidin treatment) and hesperidin (alone) treatment were observed. These proteins are involved in metabolism, muscle activity, heat shock response, redox homeostasis, protein synthesis/folding/degradation, development, ion-channel/cellular transport, and gustatory and olfactory function of the flies. Our data indicates that numerous cellular processes are involved in the temporal regulation of proteins and widespread modulations happen under rotenone treatment and, action of hesperidin could also be seen on these categories of proteins.

Biomarkers for Bone Tumors: Discovery from Genomics and Proteomics Studies and Their Challenges.

Diagnosis of bone tumor currently relies on imaging and biopsy, and hence, the need to find less invasive ways for its accurate detection. More recently, numerous promising deoxyribonucleic acid (DNA) and protein biomarkers with significant prognostic, diagnostic and/or predictive abilities for various types of bone tumors have been identified from genomics and proteomics studies. This article reviewed the putative biomarkers for the more common types of bone tumors (that is, osteosarcoma, Ewing sarcoma, chondrosarcoma [malignant] and giant cell tumor [benign]) that were unveiled from the studies. The benefits and drawbacks of these biomarkers, as well as the technology platforms involved in the research, were also discussed. Challenges faced in the biomarker discovery studies and the problems in their translation from the bench to the clinical settings were also addressed.

Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in the aqueous humour of patients with primary angle closure glaucoma - a quantitative study.

BACKGROUND: Altered levels of specific matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the aqueous humour of primary open-angle glaucoma (POAG) eyes have been described. In this study, levels of specific MMPs and TIMPs in the aqueous humour of primary angle-closure glaucoma (PACG) eyes were measured and compared with those of POAG as well as non-glaucoma control eyes. METHODS: Aqueous humour from 16 PACG, 28 POAG and 27 control eyes were sampled during intraocular surgery. Levels of total protein, MMP-2, MMP-3, TIMP-1 and TIMP-2 were quantified by protein assay and enzyme immunoassay. RESULTS: Total protein levels were significantly higher in PACG (0.426 ± 0.126 mg/ml, p = 0.043) and POAG (0.578 ± 0.360 mg/ml, p = 0.007) compared to controls (0.292 ± 0.192 mg/ml). The difference between PACG and POAG was not significant (p = 0.158). MMP-2 was significantly higher in PACG (p = 0.032) and POAG (p < 0.001) compared to controls. The difference between PACG and POAG was also not significant (p = 0.133). MMP-3 was significantly higher in POAG compared to controls (p = 0.002) and PACG (p = 0.029). The difference between PACG and controls was not significant (p = 0.962). TIMP-1 was significantly higher in PACG (p = 0.049) and POAG (p = 0.010) compared to controls. The difference between PACG and POAG was also not significant (p = 0.961). TIMP-2 was significantly higher in POAG (p = 0.004) compared to controls. The difference between PACG and either controls or POAG was not significant (p > 0.05). Although not statistically significant (p > 0.05), the MMP-2/TIMP-2 ratio was highest in PACG (2.83 ± 7.40), followed by POAG (1.38 ± 1.55) and controls (1.34 ± 3.05). Similarly, the MMP-2/TIMP-1 ratio was highest in PACG (1.50 ± 1.69), followed by POAG (1.40 ± 0.77) and controls (1.15 ± 0.92). The MMP-2 + MMP-3/TIMP-1 + TIMP-2 ratio was higher in PACG (0.83 ± 0.80) and POAG (0.82 ± 0.53) compared to controls (0.70 ± 0.63). In both POAG and PACG, there were no significant differences in the levels of total protein, MMP-2, MMP-3, TIMP-1 and TIMP-2 between patients on prostaglandin analogues and those not. CONCLUSION: We found altered levels of MMPs and TIMPs as well as imbalance of MMP:TIMP ratios in the aqueous humour of PACG eyes that were different from POAG and non-glaucoma control eyes.

Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.

Identification of potential complementary serum biomarkers to differentiate prostate cancer from benign prostatic hyperplasia using gel- and lectin-based proteomics analyses.

Diagnosis of prostate cancer (PCa) is currently much reliant on the invasive and time-consuming transrectal ultrasound-guided biopsy of the prostate gland, particularly in light of the inefficient use of prostate-specific antigen as its biomarker. In the present study, we have profiled the sera of patients with PCa and benign prostatic hyperplasia (BPH) using the gel- and lectin-based proteomics methods and demonstrated the significant differential expression of apolipoprotein AII, complement C3 beta chain fragment, inter-alpha-trypsin inhibitor heavy chain 4 fragment, transthyretin, alpha-1-antitrypsin, and high molecular weight kininogen (light chain) between the two groups of patients' samples. Our data are suggestive of the potential use of the serum proteins as complementary biomarkers to effectively discriminate PCa from BPH, although this requires further extensive validation on clinically representative populations.

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats.

BACKGROUND: Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR). RESULTS: Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril. CONCLUSION: Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension.

Salivary proteins associated with periodontitis in patients with Type 2 diabetes mellitus.

The objective of this study was to investigate the salivary proteins that are associated with periodontitis in patients with Type 2 diabetes mellitus (T2DM). Volunteers for the study were patients from the Diabetic Unit, University of Malaya Medical Centre, whose periodontal status was determined. The diabetic volunteers were divided into two groups, i.e., patients with periodontitis and those who were periodontally healthy. Saliva samples were collected and treated with 10% TCA/acetone/20 mM DTT to precipitate the proteins, which were then separated using two-dimensional polyacrylamide gel electrophoresis. Gel images were scanned using the GS-800(TM) Calibrated Densitometer. The protein spots were analyzed and expressed in percentage volumes. The percentage volume of each protein spot was subjected to Mann-Whitney statistical analysis using SPSS software and false discovery rate correction. When the expression of the salivary proteins was compared between the T2DM patients with periodontitis with those who were periodontally healthy, seven proteins, including polymeric immunoglobulin receptor, plastin-2, actin related protein 3, leukocyte elastase inhibitor, carbonic anhydrases 6, immunoglobulin J and interleukin-1 receptor antagonist, were found to be differentially expressed (p < 0.01304). This implies that the proteins may have the potential to be used as biomarkers for the prediction of T2DM patients who may be prone to periodontitis.

Identification of immunoreactive secretory proteins from the stationary phase culture of Burkholderia pseudomallei.

Bacterial secreted proteins are known to be involved in virulence and may mediate important host-pathogen interactions. In this study, when the stationary phase culture supernatant of Burkholderia pseudomallei was subjected to 2-DE, 113 protein spots were detected. Fifty-four of the secreted proteins, which included metabolic enzymes, transcription/translation regulators, potential virulence factors, chaperones, transport regulators, and hypothetical proteins, were identified using MS and database search. Twelve of these proteins were apparently reactive to antisera of mice that were immunised with B. pseudomallei secreted proteins. These proteins might be excellent candidates to be used as diagnostic markers or putative candidate vaccines against B. pseudomallei infections.

Patients with ovarian carcinoma excrete different altered levels of urine CD59, kininogen-1 and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin.

BACKGROUND: Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers for early stage detection. Proteins that are aberrantly excreted in the urine of cancer patients are excellent biomarker candidates for development of new noninvasive protocol for early diagnosis and screening purposes. In the present study, urine samples from patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis and the profiles generated were compared to those similarly obtained from age-matched cancer negative women. RESULTS: Significant reduced levels of CD59, kininogen-1 and a 39 kDa fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), and enhanced excretion of a 19 kDa fragment of albumin, were detected in the urine of patients with ovarian carcinoma compared to the control subjects. The different altered levels of the proteins were confirmed by Western blotting using antisera and a lectin that bind to the respective proteins. CONCLUSION: CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary biomarkers in the development of new noninvasive protocols for diagnosis and screening of ovarian carcinoma.

Patients with congenital hypothyroidism demonstrate different altered expression of plasma fibrinogen and haptoglobin polypeptide chains.

OBJECTIVES: To compare the plasma protein profiles of treated and untreated congenital hypothyroidism (CH) patients with those of normal control infants. DESIGN AND METHODS: Plasma samples were subjected to two-dimensional gel electrophoresis and silver staining or lectin detection. Resolved protein spots were analyzed by using computerized densitometry software. RESULTS: The significant enhanced expression of the fibrinogen gamma-chain and reduced expression of haptoglobin beta-chain were demonstrated in the profiles of untreated CH patients using both silver staining and lectin detection methods. Lectin detection resolved only a single cluster of the haptoglobin beta-chain for the untreated patients, in contrast to five isoform clusters detected in the controls' and treated CH patients' profiles. CONCLUSIONS: Plasma from untreated CH patients demonstrated different altered expression of fibrinogen and haptoglobin polypeptide chains, which was normalized when patients were treated. Our data also suggest differences in structures of the N-glycans of haptoglobin beta-chain of the untreated CH patients.

Patients with nasopharyngeal carcinoma demonstrate enhanced serum and tissue ceruloplasmin expression.

The proteomics approach was adopted to study the simultaneous expression of serum proteins in patients with nasopharyngeal carcinoma (NPC). We have subjected unfractionated whole sera of ten newly diagnosed Malaysian Chinese patients with WHO type III NPC to two-dimensional gel electrophoresis (2-DE) and image analysis. The results obtained were then compared to that generated from sera of ten normal healthy controls of the same ethnic group and range of age. Our data demonstrated that the serum high abundance 2-DE protein profiles of NPC patients were generally similar to that of the controls, with exception of the ceruloplasmin (CPL) spots (identified by mass spectrometric analysis and MASCOT database search), which showed higher expression. The enhanced expression of CPL in the patients' sera was confirmed by competitive ELISA. Immunohistochemical analysis of nasopharyngeal lesions of NPC patients demonstrated moderate to strong positive CPL staining in the cytoplasm of cells at the regions of malignancy but only weak cytoplasmic staining at normal epithelial lining areas. When follow-up 2-DE and ELISA studies were performed on five of the NPC patients who responded positively to six months treatment, the difference in CPL expression was no longer significant.

Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster.

Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription. Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock.

Revealing Glycoproteins in the Secretome of MCF-7 Human Breast Cancer Cells.

Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer.

Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins.

Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding.

Effects of Tamarindus indica fruit pulp extract on abundance of HepG2 cell lysate proteins and their possible consequential impact on metabolism and inflammation.

The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism.

Identification of immunogenic proteins from Burkholderia cepacia secretome using proteomic analysis.

Burkholderia cepacia is an opportunistic human pathogen associated with lung infections. Secretory proteins of B. cepacia are known to be involved in virulence and may mediate important host-pathogen interactions. In the present study, secretory proteins isolated from B. cepacia culture supernatant were separated using two-dimensional gel electrophoresis, followed by Western blot analysis to identify the immunogenic proteins. Mice antibodies raised to B. cepacia inactivated whole bacteria, outer membrane protein and culture filtrate antigen detected 74, 104 and 32 immunogenic proteins, respectively. Eighteen of these immunogenic proteins which reacted with all three antibodies were identified and might be potential molecules as a diagnostic marker or a putative candidate vaccine against B. cepacia infections.

Different altered stage correlative expression of high abundance acute-phase proteins in sera of patients with epithelial ovarian carcinoma.

BACKGROUND: The general enhanced expression of alpha1-antichymotrypsin (ACT), clusterin (CLU), alpha1-antitrypsin (AAT), haptoglobin beta-chain (HAP), and leucine rich glycoprotein (LRG) in the sera of patients with epithelial ovarian carcinoma (EOCa) was recently reported. In the present study, we compared the expression of the serum acute-phase proteins (APPs) in the patients according to their stages of cancer. RESULTS: Different altered stage correlative expression of the high abundance serum APPs was demonstrated in sera of the patients studied. While the expression of ACT, HAP and AAT appeared to demonstrate positive correlation with the three initial stages of the cancer, inverse correlation was apparently detected in the expression of LRG and CLU. For patients who were diagnosed with stage IV of the cancer, expression of the serum APPs did not conform to the altered progression changes. CONCLUSION: Our results highlight the potential prognostic significance of selective high abundance serum APPs in patients with EOCa.

Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: analysis using 2-DE with silver staining and lectin detection methods.

The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.

Aberrant expression of acute-phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumors.

We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.