ABSTRACT
Dendrobium Sabin Blue is an important orchid hybrid that has been grown extensively as cut flower, potted plant and is also popular for its deep purplish blue flowers. The most efficient long term conservation method of this hybrid is through cryopreservation. Cryopreservation involving the vitrification method consists of explants exposure to highly concentrated cryoprotective solution followed by freezing rapidly in liquid nitrogen. However, these treatments involved highly concentrated cryoprotectant that could incur toxicity to the explants. Hence, cryopreservation protocol requires biochemical analyses in understanding the damages or injuries occurred during cryopreservation treatments. In this study, biochemical analyses revealed a general reduction in chlorophyll, carotenoid and porphyrin content to 0.40 µg/g F W (thawing stage), 31.50 µg/g F W unloading stage and 2230.41 µg/g F W (thawing stage), respectively in comparison to the control treatments. In addition, increased level in proline content were obtained at different cryopreservation stages with highest level (5.42 µmole/g F W) recorded at the PVS2 dehydration stage. Fluctuated outcomes were obtained in catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) enzyme activities in PLBs exposed to different cryopreservation stages. Lowest values recorded for CAT enzyme activity were obtained at the dehydration stage (3.94 U/g). Lowest POX enzyme activities were obtained at the dehydration (122.36 U/g) and growth recovery (106.40 U/g) stages. Additionally, lowest APX enzyme activities values were recorded at the thawing (7.47 U/g) and unloading (7.28 U/g) stages. These have contributed to low regeneration of Dendrobium Sabin Blue protocorm like bodies (PLBs) following cryopreservation. Hence, in the future experimental design, exogenous antioxidant could be included in the cryopreservation procedures to improve the existing protocol.
ABSTRACT
Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC-ESI-MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.
Subject(s)
Proteomics/methods , Chromatography, Liquid , Entamoeba histolytica , Membrane Proteins , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa. METHODS: E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC-ESI-MS/MS and LC-MALDI-TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB). RESULTS: A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC-ESI-MS/MS and 107 proteins by LC-MALDI-TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC-ESI-MS/MS and LC-MALDI-TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts. CONCLUSION: This study showed that complementary use of LC-ESI-MS/MS and LC-MALDI-TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.
