Characterization of endoplasmic reticulum from castor bean and the cloning of a plant phosphatase: a basis for comprehensive plant organelle proteomics research

The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum

Genetic regulation of aleurone cell fate in Zea mays

The outermost layer of the endosperm is a specific cell type called the aleurone which is one of the grain quality determining factors because of its high content in lipid, minerals and high quality proteins compared to starchy endosperm. The aleurone layer is also an attractive system to study cell fate determination because of the simplicity and plasticity of aleurone cell fate. Here we report the identification of naked endosperm (nkd) genes which are involved in aleurone differentiation in maize. The nkd mutant shows defects in aleurone cell identity and has approximately 3 outer cell layers instead of the single in WT. However these outer cells do not contain dense granular cytoplasm typical of normal aleurone and have sporadic expression of a Vp1 promoter GUS transgene, which is an aleurone identity marker. The nkd mutant phenotype shows 15:1 segregation ratio in F2 populations suggesting two recessive genes are involved in this phenotype. We performed map-based cloning and found two homologous genes in syntenic regions. The INDETERMINATE1 domain containing transcription factors ZmIDDveg9 and ZmIDD9 correspond to the nkd1 and nkd2 mutant genes on chromosomes 2 and 10, respectively. Independent Ds transposon insertion alleles of nkd1 and nkd2, nkd1-Ds and nkd2-Ds respectively, failed to complement the original nkd mutant. A Nkd2-RNAi line, in which both of nkd genes were knocked down, also showed the nkd mutant phenotype. The nkd transcripts were most abundant in developing kernels around 11 to16 days after pollination. The NKD proteins have putative nuclear localization signals as other IDD genes and GFP fusion proteins showed nuclear localization. The mutant phenotype and gene expression pattern suggest NKD functions in aleurone cell fate acquisition and differentiation

Post−translational mechanisms of the ZIP family of zinc channels

Zinc is an essential trace metal involved in diverse cellular processes. Cellular zinc levels are controlled by three families of proteins, including ZIP channels, which facilitate zinc influx into the cytosol. Aberrant function of many ZIP channels has been associated with human diseases. However, their cellular mechanisms are relatively unclear. Importantly, our group have reported that ZIP7 function is triggered by CK2−mediated phosphorylation on residues S275 and S276, and have created a pZIP7 antibody that recognises this phosphorylated form of ZIP7. This project therefore aimed to decipher post−translational mechanisms of three ZIP channels: ZIP7, ZIP6, and ZIP10. Computational analysis of ZIP sequences revealed salient characteristics of ZIP channels, especially those belonging to the LIV−1 subfamily, and detected multiple potential phosphorylation sites in the cytosolic loop between TM3 and TM4 of ZIP3, ZIP4, ZIP6, ZIP7 and ZIP10. Characterisation of the pZIP7 antibody revealed that it specifically recognised ZIP7 when phosphorylated on S275 and S276 and accurately indicated increased or impaired function of the protein. Employing different antibody arrays, phosphorylation of multiple kinases by ZIP7 overexpression or zinc was demonstrated, introducing multiple signalling pathways as downstream cascades of ZIP7−mediated zinc release from intracellular stores. An investigation on ZIP6 detected that it was phosphorylated by CK2, CK1, GSK−3, and PLK1 exclusively in mitotic cells. This led to a deeper discovery of the cellular mechanism of ZIP6 in mitosis involving its heteromer formation with ZIP10 and its binding to pS727 STAT3, which also bound to pStathmin, a protein required for microtubule reorganisation. Importantly, ZIP6 and ZIP10 antibody treatment successfully inhibited mitosis in multiple breast cancer cell lines, either nocodazole−induced or endogenous. Collectively, this project has provided a deeper insight into ZIP7, ZIP6 and ZIP10 cellular mechanisms, introduced pZIP7 antibody as a potential biomarker, and proposed ZIP6 and ZIP10 antibodies as promising mitosis−blocking agents

A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties

Philosophiae Doctor - PhDThis study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.South Afric

Goat Immunity to Helminthes

Goat hematology, especially, shares considerable attention since the last 1980s. Large number of discrepant normal hematologic values is reported. The discrepancies resulted came from the differences in age group, breed, and health standing of goats. This makes it further complex with variances in climate of the region, its environment, and size and methodology applied. With time, many inconsistencies, reasonably standardization in normal caprine kinetic hematologic values, are in place. Both goats and sheep are infested by the same key digestive tract helminthes (DTHs) diseases. Helminthes are exceedingly ubiquitous worm parasites that progressed to adopt with many erudite means to evade host immune system

The phenomenon of multi-drug resistance in the treatment of malignant tumors

Multi-drug resistance (MDR) is a condition when there is broad cross-resistance of cells to various agents which are different in structure and effect. Modern perceptions on mechanisms of MDR development in malignant tumors have been considered, in particular, in tre­ating breast cancer. Physiological functions and contribution to MDR development of ABC-transporter protein families have been described. The role of activation of glutathione system enzymes and apoptosis-regulating proteins in MDR formation has been shown. Key Words: multi-drug resistance, ABC-transporters, glutathione system, apoptosis-controlling genes

Trypanosoma congolense invariant surface glycoprotein: a potential diagnostic antigen for animal African trypanosomiasis.

Master of Science in Biochemistry. University of KwaZulu-Natal, Pietermaritzburg 2016.Abstract available in PDF file