The Differential Expression of Aqueous Soluble Proteins in Breast Normal and Cancerous Tissues in Relation to Stage and Grade of Patients

Breast cancer is a leading cause of female deaths worldwide. In Malaysia, it is the most common form of female cancer while Infiltrating ductal carcinoma (IDC) is the most common form of breast cancer. A proteomic approach was used to identify changes in the protein profile of breast cancerous and normal tissues. The patients were divided into different cohorts according to tumour stage and grade. We identified twenty-four differentially expressed hydrophilic proteins. A few proteins were found significantly related to various stages and grades of IDC, amongst which were SEC13-like 1 (isoform b), calreticulin, 14-3-3 protein zeta, and 14-3-3 protein eta. In this study, we found that by defining the expression of the proteins according to stages and grades of IDC, a significant relationship between the expression of the proteins with the stage or grade of IDC can be established, which increases the usefulness of these proteins as biomarkers for IDC

THE PROTEIN PROFILING OF ASIAN GIANT TOAD SKIN SECRETIONS AND THEIR ANTIMICROBIAL ACTIVITY

Objective: The skin secretions of toads are a rich source of bioactive peptides and proteins, which offer a wide range of therapeutic application. The current study was designed to elucidate the antimicrobial activity of Bufo asper skin secretions.Methods: Proteomic analysis of electrically stimulated skin secretions were mapped using SDS-PAGE followed by LC-MS/MS. In total,>50 proteins were identified with a molecular weight ranging from 20 to 250 KDa. The antimicrobial activity was performed by an agar-well diffusion method to measure the diameter of inhibition zone (DIZ) as well as microdilution technique to determine the minimum inhibitory concentration (MIC).Results: Toad's skin secretion (TSS) exhibited broad spectrum growth inhibitory activity against both Gram-positive and Gram-negative bacteria; with more pronounce activity towards Staphylococcus aureus and Bacillus subtilis, with MIC 12.25±0.4 and 25±1.3 μg/ml, respectively. Moreover, the proteomic profile of Bufo asper skin secretion has revealed the presence of interesting proteins such as, actin, histone H4 and heat shock proteins (HSP90, HSP70 and HSC70).Conclusion: we anticipate that the collective functions of proteins and peptides with a wide range of diversity may contribute to the TSS antimicrobial activity.Keywords: Bufo asper, Skin secretion, Antimicrobial, Proteomics

Overexpression of Heat Shock Cognate Protein 71 kDa and Pyruvate Dehydrogenase in the Brain Tissue at the Early Stage of High Fat Diet Consumption

High-fat diet (HFD) increases the risk of obese, while obesity increases the prevalence of metabolic syndrome and non-communicable diseases. Therefore, it will be interesting to evaluate the changes in metabolic parameters and brain profile upon the early consumption of HFD. In this study, a total of 12 Sprague-Dawley male rats were divided into two groups (n = 6), each group was fed with normal diet and HFD (39% of total calories from fats), respectively, for 6 weeks consecutively. The body weight, blood glucose, cholesterol and triglyceride values were measured.  Furthermore, the brain and visceral adipose tissues were harvested at the end of the experiment. Protein was extracted from the brain tissue, and the protein extracts were separated by using two-dimensional gel electrophoresis and analyzed by liquid chromatography tandem mass spectrometric analysis (LC/MS/MS). In terms of food calorie, the rats fed with HFD consumed more energy than the rats fed with normal diet. Nevertheless, the blood triglyceride and cholesterol, and the visceral adipose tissue of both the HFD and normal diet fed rats were indifferent. At the molecular level, overexpression of stress proteins, namely heat shock cognate protein 71 kDa (Hsc70)  and pyruvate dehydrogenase were detected in brain tissue of HFD group. These results suggest that HFD intake causing significant change in brain proteins profile at the early phase of its consumption when no clear metabolic changes were observed. This showed that the brain was affected by HFD

Comparison of Protein Expression Profiles of Different Stages of Lymph Nodes Metastasis in Breast Cancer

Breast cancer is the most common cancer among women worldwide. Breast cancer metastasis primarily happens through lymphatic system, where the extent of lymph node metastasis is the major factor influencing staging, prognosis and therapeutic decision of the disease. We aimed to study the protein expression changes in different N (regional lymph nodes) stages of breast cancer. Protein expression profiles of breast cancerous and adjacent normal tissues were mapped by proteomics approach that comprises of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry (LC-MS/MS) analysis. Calreticulin and tropomyosin alpha 3 chains were the common up-regulated proteins in N0, N1 and N2 stages of breast cancer. Potential biomarker for each N stage was HSP 70 for N0, 80 k protein H precursor and PDI for N1 stage while 78 kDa glucose-regulated protein was found useful for N2 stage. In addition, significant up-regulation of PDI A3 was detected only in the metastasized breast cancer. The up-regulation expression of these proteins in cancerous tissues can potentially use as indicators for diagnosis, treatment and prognosis of different N stages of breast cancer

Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris

Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS–PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72 h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection

SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05) at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively