Protein solubility is a critical prerequisite to any proteomics analysis. Combination of urea/thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have been routinely used to enhance protein solubilization for oil palm proteomics studies in recent years. The goals of these proteomics analysis are essentially to complement the knowledge regarding the regulation networks and mechanisms of the oil palm fatty acid biosynthesis. Through omics integration, the information is able to build a regulatory model to support efforts in improving the economic value and sustainability of palm oil in the global oil and vegetable market. Our study evaluated the utilization of sodium deoxycholate as an alternative solubilization buffer/additive to urea/thiourea and CHAPS. Efficiency of urea/thiourea/CHAPS, urea/CHAPS, urea/sodium deoxycholate and sodium deoxycholate buffers in solubilizing the oil palm (Elaeis guineensis var. Tenera) mesocarp proteins were compared. Based on the protein yields and electrophoretic profile, combination of urea/thiourea/CHAPS were shown to remain a better solubilization buffer and additive, but the differences with sodium deoxycholate buffer was insignificant. A deeper mass spectrometric and statistical analyses on the identified proteins and peptides from all the evaluated solubilization buffers revealed that sodium deoxycholate had increased the number of identified proteins from oil palm mesocarps, enriched their gene ontologies and reduced the number of carbamylated lysine residues by more than 67.0%, compared to urea/thiourea/CHAPS buffer. Although only 62.0% of the total identified proteins were shared between the urea/thiourea/CHAPS and sodium deoxycholate buffers, the importance of the remaining 38.0% proteins depends on the applications. The only observed limitations to the application of sodium deoxycholate in protein solubilization were the interference with protein quantitation and but it could be easily rectified through a 4-fold dilution. All the proteomics data are available via ProteomeXchange with identifier PXD013255. In conclusion, sodium deoxycholate is applicable in the solubilization of proteins extracted from oil palm mesocarps with higher efficiency compared to urea/thiourea/CHAPS buffer. The sodium deoxycholate buffer is more favorable for proteomics analysis due to its proven advantages over urea/thiourea/CHAPS buffer.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample.
Royal jelly is widely consumed in the community and has perceived benefits ranging from promoting growth in children and improvement of general health status to enhancement of longevity for the elderly. However, royal jelly consumption has been linked to contact dermatitis, acute asthma, anaphylaxis and death. High prevalence of positive skin tests to royal jelly have been reported among atopic populations in countries with a high rate of royal jelly consumption. The present study is aimed to identify the major allergens of royal jelly. Royal jelly extract was separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional electrophoresis (2-D). Immunoblotting of the SDS-PAGE and 2-D profiles were performed to identify the allergenic spots. Spots were then excised from the 2-D gel, digested with trypsin and analyzed by mass spectrometry. The SDS-PAGE of royal jelly extract revealed 18 bands between 10 to 167 kD. Western blot of the fractionated proteins detected 15 IgE-binding bands between 14 to 127 kD with seven major allergens of 32, 40, 42, 49, 55, 60 and 67 kD using serum from 53 subjects with royal jelly allergy. The 2-D gel fractionated the royal jelly proteins to more than 50 different protein spots. Out of these, 30 spots demonstrated specific IgE affinity to the sera tested. Eight spots of the major royal jelly allergens were selected for mass-spectrometry analysis. Digested tryptic peptides of the spots were compared to the amino acid sequence search in protein databases which identified the fragments of royal jelly homologus to major royal jelly protein 1 (MRJ1) and major royal jelly protein 2 (MRJ2). In conclusion, the major allergens of royal jelly are MRJ1 and MRJ2 in our patients' population.
Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.
Children with heavy Trichuris infestation were compared with paediatric amoebic dysentery patients and normal children. Heavy Trichuris infestation was diagnosed by visualization of worms on anoscopy. Patients with heavy Trichuris infection had a longer duration of disease, more frequent hospitalization and a higher rate of rectal prolapse than did patients with amoebiasis. Five Trichuris children also had clubbing. Trichuris patients had lower mean haematrocrits (27%) and serum albumin (3-3 gm%) than did patients with amoebiasis (32% and 3-7 gm% respectively). Coinfection with Shigella and Salmonella was significantly increased in patients with heavy Trichuris infection compared to both amoebic and control group children. Trichuris patients were infected with Entamoeba histolytica more frequently (46%) than normal children. Heavy Trichuris infection is the probable cause of symptoms and signs seen in these patients.
Wax apple is one of the underutilized fruits that is considered a good source of fibers, vitamins, minerals as well as antioxidants. In this study, a comparative analysis of the developments of wax fruit ripening at the proteomic and metabolomic level was reported. 2D electrophoresis coupled with MALDI-TOF/TOF was used to compare the proteome profile from three developmental stages named immature, young, and mature fruits. In general, the protein expression profile and the identified proteins function were discussed for their potential roles in fruit physiological development and ripening processes. The metabolomic investigation was also performed on the same samples using quadrupole LC-MS (LC-QTOF/MS). Roles of some of the differentially expressed proteins and metabolites are discussed in relation to wax apple ripening during the development. This is the first study investigating the changes in the proteins and metabolites in wax apple at different developmental stages. The information obtained from this research will be helpful in developing biomarkers for breeders and help the plant researchers to avoid wax apple cultivation problems such as fruit cracking.
Synovial sarcoma (SS) is a malignant soft tissue tumour of uncertain histogenesis which is defined by the translocation t(X;18) that produces the fusion oncogenes SYT-SSX. The emergence of transducer-like enhancer of split 1 (TLE1) as a new immunohistochemical (IHC) marker for SS has offered an alternative to pathologists in differentiating SS from other histological mimics, especially in the setting of limited molecular facilities. We investigated the utility of IHC TLE1 expression against histomorphological features and other IHC markers in SS and non-SS tumours. Twenty-six cases of histologically diagnosed SS and 7 non-SS (for which SS was in the differential diagnosis) were subjected to TLE1 IHC staining, which was graded from 0 to 3+. Of the 26 SS cases, 12 each were biphasic and monophasic types and 2 were poorly-differentiated. TLE1 was expressed in 22/26 (84.6%) SS cases, of which 11/12 (91.7%) were biphasic, 10/12 (83.3%) monophasic and 1/2 (50%) poorly-differentiated tumours. Two of 7 (28.6%) non-SS cases were positive for TLE1. Immunopositivity of SS and non-SS cases for EMA were 20/26 (76.9%) and 2/7 (28.6%) respectively and for CK7 were 7/26 (26.9%) and 0/7 (0%) respectively. All cases were negative for CD34. Consistent histomorphological features for SS included mild nuclear pleomorphism, alternating tumour cellularity, fascicular growth pattern and thick ropy stromal collagen. In conclusion, TLE1 is not a stand-alone diagnostic IHC marker for SS. However, in the absence of molecular studies, it can contribute added diagnostic value in combination with morphological evaluation and other IHC markers such as EMA and CD34.
The haemolysins produced by Aeromonas species were detected and compared by two assay methods--a modified blood agar plate assay and the rabbit erythrocyte haemolysin method. Both assays showed a high level of agreement (86%). The titres of the rabbit erythrocyte haemolysin assay correlated with the haemolytic zone diameter of the ox blood agar assay. In addition the agar haemolysin assay had simple media requirements, was easy to perform and results were well defined.
The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.
Recently, chitin and chitosan are widely investigated for food preservation and active packaging applications. Chemical, as well as biological methods, are usually adopted for the production of these biopolymers. In this study, modification to a chemical method of chitin synthesis from shrimp shells has been proposed through the application of high-frequency ultrasound. The impact of sonication time on the deproteinization step of chitin and chitosan preparation was examined. The chemical identities of chitin and chitosan were verified using infrared spectroscopy. The influence of ultrasound on the deacetylation degree, molecular weight and particle size of the biopolymer products was analysed. The microscopic characteristics, crystallinity and the colour characteristics of the as-obtained biopolymers were investigated. Application of ultrasound for the production of biopolymers reduced the protein content as well as the particle size of chitin. Chitosan of high deacetylation degree and medium molecular weight was produced through ultrasound assistance. Finally, the as-derived chitosan was applied for beef preservation. High values of luminosity, chromatid and chrome were noted for the beef samples preserved using chitosan films, which were obtained by employing biopolymer subjected to sonication for 15, 25 and 40 min. Notably; these characteristics were maintained even after ten days of packaging. The molecular weight of these samples are 73.61 KDa, 86.82 KDa and 55.66 KDa, while the deacetylation degree are 80.60%, 92.86% and 94.03%, respectively; in the same order, the particle size of chitosan are 35.70 μm, 25.51 μm and 20.10 μm.
The main challenge in microfiltration (MF) is membrane fouling, which leads to a significant decline in permeate flux and a change in membrane selectivity over time. This work aims to elucidate the mechanisms of membrane fouling in cold MF of skim milk by identifying and quantifying the proteins and minerals involved in external and internal membrane fouling. Microfiltration was conducted using a 1.4-μm ceramic membrane, at a temperature of 6±1°C, cross-flow velocity of 6m/s, and transmembrane pressure of 159kPa, for 90min. Internal and external foulants were extracted from a ceramic membrane both after a brief contact between the membrane and skim milk, to evaluate instantaneous adsorption of foulants, and after MF. Four foulant streams were collected: weakly attached external foulants, weakly attached internal foulants, strongly attached external foulants, and strongly attached internal foulants. Liquid chromatography coupled with tandem mass spectrometry analysis showed that all major milk proteins were present in all foulant streams. Proteins did appear to be the major cause of membrane fouling. Proteomics analysis of the foulants indicated elevated levels of serum proteins as compared with milk in the foulant fractions collected from the adsorption study. Caseins were preferentially introduced into the fouling layer during MF, when transmembrane pressure was applied, as confirmed both by proteomics and mineral analyses. The knowledge generated in this study advances the understanding of fouling mechanisms in cold MF of skim milk and can be used to identify solutions for minimizing membrane fouling and increasing the efficiency of milk MF.
The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.
Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.
Matched MeSH terms: Recombinant Fusion Proteins/analysis; Green Fluorescent Proteins/analysis