Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZPss.

HrpZ-a harpin from Pseudomonas syringae-is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities

A comparative study on dismissal by operation of law in terms of the Public Service Act: South Africa and Namibia

Magister Philosophiae - MPhilThe right to fair dismissal in South Africa is prescribed in the Labour Relation Act 66 of 1995 as amended. Employees may only be dismissed on grounds of misconduct, incapacity and operational requirements. The requirements for dismissal of employees based on misconduct and incapacity are further addressed in Schedule 8 to the LRA, the Code of Good Practice: Dismissal. Dismissal for misconduct needs to be fair in terms of both procedure and substance. Procedural fairness generally involves holding a disciplinary hearing before dismissing an employee. In terms of the South African Public Service Act 103 of 1994 as amended, an employee who absents him-/herself from official duties without permission of his or her head of department, office or institution for a period exceeding one calendar month is deemed dismissed by operation of law. Employees dismissed as such by operation of law in terms of the PSA are therefore not afforded the right to appear in the disciplinary hearing as provided for in the LRA. South African courts have dealt with a number of cases relating to dismissal by operation of law in the public service. Some of the employees dismissed were reinstated by the courts. Reasons for reinstatement included failing to meet the jurisdictional requirements before invoking dismissal by operation of law. The research will attempt to clarify the substantive and procedural steps required to render a dismissal by operation of law in terms of the PSA fair in South Africa

Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings

The enzyme quinoprotein glucose dehydrogenase (GDH) catalyses the oxidation of glucose to gluconic acid by direct oxidation in the periplasmic space of several Gram-negative bacteria. Acidification of the external environment with the release of gluconic acid contributes to the solubilization of the inorganic phosphate by biofertilizer strains of the phosphate-solubilizing bacteria. Glucose dehydrogenase (gcd) gene from Escherichia coli, and Azotobacter-specific glutamine synthetase (glnA) and phosphate transport system (pts) promoters were isolated using sequence-specific primers in a PCR-based approach. Escherichia coli gcd, cloned under the control of glnA and pts promoters, was mobilized into Azotobacter vinelandii AvOP and expressed. Sorghum seeds were bacterized with the transgenic azotobacters and raised in earthen pots in green house. The transgenic azotobacters, expressing E. coli gcd, showed improved biofertilizer potential in terms of mineral phosphate solubilization and plant growth-promoting activity with a small reduction in nitrogen fixation ability

Overlapping sets of transcripts from host and non-host interactions of tomato are expressed early during non-host resistance

Natural immunity present in all the plants against most of the pathogens is called as non-host resistance (NHR). Although NHR is most durable form of resistance, it was less studied compared to other forms of resistance. We compared transcriptional changes in tomato during non-host (Magnaporthe grisea) and compatible (Alternaria alternata f. sp. lycopersici) interactions using Agilent microarray GeneChip containing ~44,000 probe sets. The experiment was designed to understand the early and late responses of tomato leaves inoculated with non-host and compatible pathogens. Microarray data revealed that the expression profiles in the non-host and compatible interactions at 6 h post inoculation (hpi) and 24 hpi largely overlapped indicating that a set of genes are activated during plant-pathogen interaction. However, these genes were expressed much earlier in NHR compared to a compatible interaction. NHR is, therefore, an accelerated and amplified basal defense response. Transcripts involved in energy production (carbohydrate metabolism and photosynthesis) were down-regulated, whereas transcripts associated with catabolic processes (starch and sucrose hydrolysis) were up-regulated in both the interactions at 6 and 24 hpi. We have also identified that the pathway involved in synthesis of volatile compounds like 2-phenylethanol was induced during NHR in tomato. This is the first report of transcriptome profile in tomato during non-host interactions against M. grisea

Electrochemical studies of the ligand 1-hydroxyl-3-aminopropilydenephosphonic acid (APD) towards bone cancer therapy

The stability constants for the ligand 1-hydroxyl-3-aminopropilydene diphosphonic acid (APD) or Pamidronate with metal ions CdII, PbII and ZnII were established in this work by sampled direct current polarography (DCTAST). Due to precipitation of the metal-ligand complexes in the pH range about 4.0 to 5.0 at typical glass electrode potentiometric conditions, these systems could not be studied by glass electrode potentiometry (GEP). The concept of Virtual Potentiometry (VP) was used in the modelling of the metal-ligand system and refinement of stability constants to evaluate further the metal-ligand models derived from DCTAST. Virtual potentiometry uses virtual potentials to refine polarographic data by employing dedicated potentiometric software, ESTA. The structure of the metal complexes determined in this work is also proposed and compared to the reported crystal structures of the metal complexes of the ligand APD. The Linear Free Energy Relationship, LFER (log KML′ vs. log KM(OH) ) for the ligand APD is derived here for the first time using the log KML′ values from literature as well as the values for CdII, PbII and ZnII determined in this work. The log KML′ values of 153SmIII–APD and 166HoIII–APD, which cannot be determined by these two techniques (GEP and DCTAST), were predicted in this work using the LFER methodology.Dissertation (MSc (Chemistry))--University of Pretoria, 2006.ChemistryMScUnrestricte

Pathogen-induced expression of harpinPss increases resistance in tobacco against fusarium oxysporum f. sp. nicotianae

HarpinPss (encoded by the hrpZ gene), a proteinaceous elicitor produced by Pseudomonas syringae pv. syringae, induces cell death in plants through hypersensitive response (HR). With an aim to generate transgenic tobacco resistant to fungal diseases, hrpZ was expressed in a secretable form, tagged with the signal peptide (SP) of PR1a, under the constitutive 35S promoter (P35S) or pathogen-inducible promoters (PIPs) like phenylalanine ammonia lyase (PAL), osmotin (OSM), and hypersensitive-related (HSR) promoters. The constitutive expression of the secretable form of hrpZ did not permit regeneration of transformed cells due to harpinPss-induced cell death. Transformants were recovered at a low frequency (2-6%) from leaf discs infected with Agrobacterium harbouring the SP-hrpZ driven by PIPs due to wound-induced leaky expression of harpinPss. The transgenic lines were confirmed by PCR using transgene-specific primers for SP-hrpZ. The expression of hrpZ under PIPs in transgenic lines was confirmed by Western blotting after challenging the leaves with Fusarium oxysporum f. sp. nicotianae. RT-PCR analysis also confirmed the expression of SP-hrpZ driven by PIPs in transgenic tobacco upon infection with F. oxysporum f. sp. nicotianae. The expression of harpinPss in these transgenic lines was accompanied by expression of defense-response genes such as PR1, PR2, PR3, HSR and HIN1. Transgenic tobacco plants showed enhanced resistance to F. oxysporum f. sp. nicotianae. Our findings suggest the potential use of an elicitor gene (hrpZ), driven by PIPs (PAL, OSM, and HSR) for the development of resistant plants

Root exudate-induced alterations in Bacillus cereus cell wall contribute to root colonization and plant growth promotion

The outcome of an interaction between plant growth promoting rhizobacteria and plants may depend on the chemical composition of root exudates (REs). We report the colonization of tobacco, and not groundnut, roots by a non-rhizospheric Bacillus cereus (MTCC 430). There was a differential alteration in the cell wall components of B. cereus in response to the REs from tobacco and groundnut. Attenuated total reflectance infrared spectroscopy revealed a split in amide I region of B. cereus cells exposed to tobacco-root exudates (TRE), compared to those exposed to groundnut-root exudates (GRE). In addition, changes in exopolysaccharides and lipid-packing were observed in B. cereus grown in TRE-amended minimal media that were not detectable in GRE-amended media. Cell-wall proteome analyses revealed upregulation of oxidative stress-related alkyl hydroperoxide reductase, and DNA-protecting protein chain (Dlp-2), in response to GRE and TRE, respectively. Metabolism-related enzymes like 2-amino-3-ketobutyrate coenzyme A ligase and 2-methylcitrate dehydratase and a 60 kDa chaperonin were up-regulated in response to TRE and GRE. In response to B. cereus, the plant roots altered their exudate-chemodiversity with respect to carbohydrates, organic acids, alkanes, and polyols. TRE-induced changes in surface components of B. cereus may contribute to successful root colonization and subsequent plant growth promotion

Improvement of cattle oocyte retrieval techniques and hormonal influence on in vitro embryonic development

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattl

Improvement of cattle oocyte retrieval techniques and hormonal influence on in vitro embryonic development

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattl