Browsing by Department "Department of Molecular and Cell Biology"

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    A deletion and point mutation study of the human papillomavirus type 16 major capsid gene
    (2006) Varsani, Arvind; Williamson, Anna-Lise; Jaffer, Mohamed A; Rybicki, Edward P
    Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A→T mutation at residue 266 (A266T), and of a C→G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428–483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2–9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428–465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.
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    A small-scale RNA isolation protocol useful for high-throughput extractions from recalcitrant plants
    (2010) Smart, Mariette; Roden, Laura Catherine
    Many plants indigenous to South Africa are rich in secondary and oxidizing compounds such as pigments, complex polysaccharides and polyphenols. This makes isolation of high quality RNA for analysis of gene expression difficult. Here we describe a cost-effective isolation protocol suitable for RNA extraction from recalcitrant plant species. This method uses small amounts of tissue, so is useful when material is limited, and is easy to process large numbers of samples at once. We have used the method successfully with mature leaves of Protea hybrid ‘Sylvia’, and species P. repens, Leucospermum hybrid ‘Succession’, resurrection plants Xerophyta humilis and Craterostigma pumilum, and mature needles of Pine (Pinus radiata). RNA was analyzed spectrophotometrically and was found to be of high purity with low levels of contaminating compounds. Electrophoretic analyses on denaturing formaldehyde agarose gels and an Agilent 2100 Bioanalyzer confirmed the presence of RNA of high integrity. This is the first description of plant RNA integrity number (RIN) values for these plants using the algorithm designed for analyses of plant RNA containing multiple ribosomal bands. The RNA could successfully be used for reverse transcription and gene amplification.
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    Open Access
    Abrogation of contaminating RNA activity in HIV-1 Gag VLPs
    (BioMed Central Ltd, 2011) Valley-Omar, Ziyaad; Meyers, Ann; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward
    BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/mug Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.
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    Actinomycete biodiversity assessed by culture-based and metagenomic investigations of three distinct samples in Cape Town, South Africa
    (2011) Davids, Muhammad Saeed; Meyers, Paul
    All of the samples used for actinobacterial isolation were subjected to a culture-independent (metagenomic) study. The results provided an explanation for why no actinomycetes were found in the aquatic samples, as all of the sequenced clones were shown to be most closely related to uncultured bacteria. In the terrestrial sample, a total of 120 clones were obtained and all were sent for sequencing.
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    Activation of seed-specific genes in leaves and roots of the desiccation tolerant plant, Xerophyta humilis
    (2008) Walford, Sally-Ann; Illing, Nicola; Farrant, Jill M; Denby, Katherine J
    The ability of tissues to survive almost complete loss of cellular water is a trait found throughout the plant kingdom. While this desiccation tolerance is common in seeds of most angiosperms it is rare in their vegetative tissues. Xerophyta humilis (Bak.) Dur and Schintz belongs to a small group of resurrection angiosperms and it possesses the ability to withstand extreme desiccation of greater than 90% in both its seeds and vegetative tissues and return to active metabolism upon rehydration. We have tested the hypothesis that vegetative desiccation tolerance in angiosperms has evolved as an adaptation of seed desiccation tolerance.
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    The amino acid sequence of chicken histone F3
    (1973) Brandt, Wolf Friedrich; Von Holt, Claus
    Histone F3 (III) from chicken erythrocytes was isolated by selective extraction from nucleoprotein with ethanolic-HCl and purified by a single gel filtration step. This protein was found to be homogeneous by the following criteria: gel filtration, electrophoretic mobility, N- and C-terminal amino acid residues and amino acid analysis. The primary structure of this histone was established without resorting to the use of overlapping sequences. This has been achieved with specific chemical cleavages rather than enzymatic degradations chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides, to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the N- and C-terminal amino acids in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradation of automatic sequencing, allowed a rapid and relatively nonlaborious primary structure determination. Finally, the amino acid sequence is compared with those of protamines and other histones. The evolution and the structure of this protein in relation to DNA is briefly considered.
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    An H5N1 influenza DNA vaccine for South Africa
    (2013) Mortimer, Elizabeth; Hitzeroth, Inga I; Buys, Amelia; Mbewana, Sandiswa; Rybicki, Edward P
    The highly pathogenic avian influenza virus H5N1 is a potent pandemic threat because of its frequent transmission from birds to humans and the increasing possibility of human to human transmission. During the 2009 H1N1 pandemic it was clear that rapid influenza vaccine production is a problem worldwide. Additionally, developing countries like South Africa generally cannot produce their own influenza vaccines because the traditional egg-based vaccine production method currently employed is too lengthy and too difficult to establish. As part of an exercise aimed at exploring the feasibility of producing emergency response influenza vaccines, we investigated an experimental DNA vaccine to the H5N1 influenza virus. We focused on the virion haemagglutinin, because it elicits the primary neutralising immune response following infection. Accordingly, we developed an H5N1 DNA vaccine with full-length and truncated versions of the haemagglutinin gene, to match previously developed protein candidates. Vaccinated mice developed a strong antibody response to the haemagglutinin protein. In addition, the full-length H5 gene elicited high haemagglutination inhibition titres in mice, indicating that it has potential as a candidate pandemic vaccine for South Africa.
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    An investigation into the role of cytosolic free Ca2+ in Salicylic acid mediation of disease resistance in Arabidopsis
    (2001) Petersen, Lindsay Natalie; Denby, Katherine J
    Salicylic acid (SA) accumulation upon pathogen attack is a fundamental requirement for the activation of numerous plant defence mechansms. Cytosolic free Ca2+ ([Ca2+]c) is a ubiquitous signalling molecule involved in a host of cellular processes. Using transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin, we previously reported a rapid and transient increase in [Ca2+]c upon application of exogenous SA. We now investigated the characteristics of the SA-induced [Ca2+]c increase and report that the majority of the response is derived from internal stores. It appears likely that SA triggers Ca2+-induced Ca2+-release. Preliminary evidence suggests a role for the SA-induced [Ca2+]c increase in the regulation of NPR1 expression since modulation of the SA-induced [Ca2+]c response with the extracellular Ca2+ chelator BAPTA causes a reduction in NPR1 mRNA levels. We have isolated two putative mutants that are defective in their ability to produce a SA-induced [Ca2+]c increase. Characterisation of these mutants is underway and will prove invaluable in identifying the components or events that cause the SA-induced [Ca2+]c transient, thereby aiding in the understanding of the role of [Ca2+]c in SA-mediated signal transduction.
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    An investigation of the role of Arabidopsis thaliana plant natriuretic peptide in planta
    (2009) Donaldson, Lara Elizabeth; Denby, Katherine; Gehring, Chris; Ingle, Rob
    The sessile nature of plants demands that they respond appropriately to changes in their environment (stresses) in order to survive. Critical to survival is the maintenance of water and ion homeostasis. The mechanisms by which plants achieve this are poorly understood. Traditionally plant stress responses were thought to be communicated by five classical plant hormones - auxin, cytokine, gibberellic acid, absisic acid and ethylene. Nowadays a plethora of other molecules are known to fulfil this function including nitric oxide, salicylic acid, jasmonic acid, brassinosteroids and peptide hormones. Plant natriuretic peptides have been proposed to be peptide hormones involved in maintaining water and ion homeostasis in plants. Evidence for this has been provided by studies of plant responses to exogenous natriuretic peptide treatment, however a demonstration of their function in planta remains outstanding. This study was undertaken to gain insight into the mechanisms regulating water and ion homeostasis in Arabdopsis by examining second messenger responses to stresses that perturb water and ion homeostasis; characterization of an Arabidopsis thaliana plant natriuretic peptide (atpnp-a) mutant and transcriptome analysis of AtPNP-A, in order to establish whether AtPNP-A plays a role in maintaining water and ion homeostasis in planta. Results indicated that recombinant AtPNP-A induces second messenger responses reminiscent of the response to NaCl, suggesting that AtPNP-A may play a signalling role in response to disturbances in water and ion homeostasis. In support of this, characterization of an atpnp-a mutant revealed that AtPNP-A is likely to be involved in processes that require adjustments to water and ion homeostasis including cell expansion, stomatal opening and NaCl and osmotic stress responses, consistent with reported responses to natriuretic peptide treatment. Furthermore, the atpnp-a mutant revealed a role for AtPNP-A in the defence response. Evidence to support this came from the computational analysis of AtPNP-A expression which correlates with genes involved in the defence response. Additionally, the transcriptome response to recombinant AtPNP-A treatment further implicated the involvement of AtPNP-A in the defence response. Therefore AtPNP-A is hypothesized to play a role in growth, abiotic and biotic stress responses that enables the plant to mount an integrated response to the environment.
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    Analysis of Actinobacterial Biodiversity in Marine Sediment from Gericke's Point (South Africa) and Screening of Isolates for Novel Antimycobacterial Compounds
    (2022) Dreyer, Ashleigh; Meyers, Paul
    Thirty-three (33) presumptive actinobacterial strains were isolated using traditional culturebased techniques from sediment taken from marine habitats (a subtidal zone, a rock pool and a beach area) at Gericke's Point (Garden Route National Park, Sedgefield, South Africa). Twenty-seven (27) of the 33 presumptive actinobacterial isolates were identified to the genus level: 26 Streptomyces strains and one Nocardia strain. The partial 16S-rRNA gene sequences obtained for each confirmed actinobacterial isolate were used to determine their phylogenetic positions within their respective genera. Further investigation of specific isolates was done utilising the gyrB gene to determine whether these isolates are clones. Metagenomic data generated from next-generation sequencing of 16S-rRNA amplicons were used to reveal the actinobacterial biodiversity of the Gericke's Point sediment that was not seen in the culture-dependent part of this study. A total of 1 541 544 actinobacterial partial 16S-rRNA gene sequences were identified using the SILVA 16S-rRNA gene database. Actinobacteria that could not be assigned to a class or order made up ~41% of the total actinobacterial strains found in the Gericke's Point sediment samples. The rest of the identified actinobacterial strains belonged to the orders Candidatus Microtrichales (~45%), Candidatus Actinomarinales (~9%), Propionibacteriales (~3%) and other actinobacterial orders that each made up less than one percent (<1%) of the actinobacterial strains found in Gericke's Point. The other actinobacterial orders include Bifidobacteriales, Euzebyales, Frankiales, Geodermatophilales, Micrococcales, Micromonosporales, Mycobacteriales, Pseudonocardiales, Streptomycetales and Streptosporangiales. This is one of the first detections of Frankiales strains in a marine environment. The majority (99%) of actinobacterial strains identified at Gericke's Point could not be assigned to a known genus. This represents an abundance of novel actinobacterial diversity that has yet to be revealed. Multidrug-resistance in Mycobacterium tuberculosis is a global threat to public health which has increased the need for new antibiotics to treat tuberculosis. In this study, all confirmed actinobacterial isolates and two presumptive actinobacterial isolates (29 strains in total) were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard agar overlay method. To investigate their spectrum of antibiotic activity, all isolates were also screened for activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Twenty-one (21) isolates (20 Streptomyces strains and one unidentified strain) displayed strong to very strong antimycobacterial activity (defined as a zone of growth inhibition of over 2000 mm2 ). In addition, two Streptomyces strains displayed strong to very strong activity against S. aureus ATCC 25923. Compounds that displayed strong antimycobacterial activity were analysed using High Performance Liquid Chromatography-Mass Spectrometry and resulting mass spectra were compared to those of known compounds within the Global Natural Products Social Molecular Networking (GNPS) database. Eighteen (18) strains produced compounds with no matches in the GNPS database indicating these compounds could be novel. One strain produced a potential analogue of abyssomicin L (a rare antibiotic). Overall, the results obtained in this study emphasize the potential of marine environments as a source of novel actinobacteria and novel bioactive compounds.
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    Analysis of actinobacterial biodiversity in reservoir sediment and cave soil and screening of isolates for antimycobacterial activity
    (2020) Rakiep, Adeebah; Meyers, Paul
    A total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL).
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    The analysis of an enzyme (Ce1A) and a gene system (abg) involved in the utilization of lignocellulose in the rumen
    (1996) Brown, Gordon Douglas; Thomson, Jennifer Ann
    As lignocellulose represents an abundant renewable resource, research is in progress to obtain a better understanding of the natural mechanisms whereby this resource is utilised. Of particular interest is the degradation of forage in the rumen and one research goal is to ultimately increase animal productivity through an improvement in lignocellulose utilisation. However, although the mechanisms behind lignocellulose utilisation are reasonably well understood, relatively little is known about the mechanisms which occur in the rumen. Thus, the aim of this thesis was to gain more insight into the mechanisms of lignocellulose utilisation which occur in the rumen. Initially this research was focused on the poorly characterised exo-acting cellulases from rumen bacteria. Preliminary enzymology studies on one cellulase from Ruminococcus flavefaciens FD-1, previously isolated in this laboratory, indicated that an exo-acting cellodextrinase, CelA, had been isolated. In this report, the enzyme was purified and biochemically characterised and was shown to be an exo-acting cellodextrinase.
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    Analysis of genes and enzymes involved in the degradation of cellulose and proteins by Butyrivibrio fibrisolvens H17c
    (1990) Berger, Eldie; Woods, David R
    Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate.
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    Analysis of genes and enzymes involved in the degradation of hemicellulose and cellulose by Butyrivibrio fibrisolvens H17c
    (1992) Lin, Long-Liu; Thomson, Jennifer Ann
    B. fibrisolvens H17c is a Gram-positive obligate anaerobe which has been found in the rumen of most ruminants. Strains of B. fibrisol vens have been reported to exhibit activity toward cellulosic and hemicellulosic substrates. The aim of this thesis was to screen a genebank of B. fibrisolvens H17c DNA and to isolate genes expressing cellulase and xylanase activity. Two genes encoding β-1-4- glucosidase (BglA) and endo-β-1-4-xylanase (XynB) were cloned in E. coli.
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    Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa (Baker) and its response to dehydration stress
    (2009) Abdalla, Kamal O; Rafudeen, Mohamed S
    Xerophyta viscosa Baker (family Velloziaceae) can survive extremes of dehydration (desiccation), down to 5% relative water content (RWC) and resumes full physiological activity within 80 h of rehydration. A thorough understanding of this phenomenon may provide further insight into possible mechanisms for improving drought tolerance in other plants. In this respect a comprehensive analysis of the nuclear proteome of this plant and its response to dehydration stress at 35% RWC was carried out. The RWC at 35% represents a distinct phase of the dehydration process where induction of late protection mechanisms is initiated and is a characteristic of desiccation tolerant species. We optimized nuclei isolation and nuclear protein extraction protocols and successfully employed these protocols to isolate highly purified nuclei and subsequently nuclear proteins from fully hydrated and dehydrated X. viscosa leaf samples. The integrity of the purified nuclei was confirmed with light and fluorescent microscopy. The nuclei were uniform spheres, approximately 5 μm in size. The purity and enrichment of the nuclear proteins were confirmed by chlorophyll assay and Western blot analysis. The nuclear proteins were investigated using two-dimensional (2D) and isobaric tags for relative and absolute quantitation (iTRAQ) technologies. Using the 2DE approach, a total of 438 proteins spots were reproducibly detected and analysed of which 18 protein spots were shown to be up-regulated in response to dehydration. These proteins contained both regulatory and functional proteins. The largest category comprised five novel protein factors and two proteins with unassigned functions. The second category comprised proteins involved in gene regulation and signal transduction. The third category comprised stress responsive proteins with chaperone type activities. Other categories include proteins involved in energy metabolism, protein degradation and translation. These results demonstrate that dehydration was controlled by multiple genes within the plant nucleus and X. viscosa may possess its own specific nuclear proteins that are involved in desiccation stress. In addition we comprehensively analyzed the nuclear proteome of X. viscosa using iTRAQ with two-dimensional liquid chromatography and tandem mass spectrometry to complement the data obtained from the 2DE approach. Using iTRAQ, we reproducibly University of Cape Town identified 128 proteins with confidence ¥ 95% (Ï < 0.05). Sixty six percent of the identified proteins showed consistent expression levels. The remaining 34% proteins showed significant changes in expressions. Of the latter, 23% were shown to be up regulated in response to dehydration stress. The remaining 11% were shown to be down regulated. The nuclear proteins of X. viscosa up-regulated in response to dehydration stress showed a coordinated response involving both regulatory and functional proteins and were implicated in diverse cellular functions. The characteristic feature of the X. viscosa nuclear proteins is the high level of stress molecules among the dehydration responsive proteins with evident functions in defense mechanisms compared to down regulated proteins and proteins showing consistent expression levels. These results demonstrate that enhanced defense capacity is crucial to desiccation tolerance and strongly support the notion that late dehydration responsive proteins are involved in protection of the cellular structures during dehydration. Proteins showing consistent expression levels during dehydration most likely maintain the minimum viability in cells under all conditions or may be indirectly associated with desiccation tolerance. Down-regulated proteins are likely important for plant survival under normal growth conditions. The proteins up-regulated in response to dehydration stress were assumed to be associated directly with the acquisition of desiccation tolerance. The up-regulated proteins were further categorized into nine functional groups to gain more insight into their roles in desiccation tolerance. The largest group was shown to be involved in gene regulation and signal transduction (36%), which reflects the role of the nucleus in gene expression and regulation. The second group included stress responsive molecules such as antioxidants, molecular chaperones and compatible solutes (33%). This reflects the importance of strong defense systems in preventing lipid peroxidation, protein aggregation, membrane leakage and maintaining the integrity of cellular structures during dehydration and in the dried state. The third group contained proteins involved in nucleocytoplasmic transport (10%). This might reflect the capacity of this plant to control the movement of molecules to and from the nucleus during dehydration and the importance of this process in adaptation to dehydration stress. The fourth group contained proteins involved in protein translation (7%). Proteins categorized to other functions, include proteins with miscellaneous and unknown functions. Proteins with unknown functions were considered to be X. viscosa nuclear-specific proteins. There was good correlation between the up-regulated proteins identified by 2-DE and iTRAQ approaches. In conclusion, this study revealed that X. viscosa nuclear proteome was responsive to dehydration stress and desiccation tolerance is University of Cape Town genetically encoded. Secondly, X. viscosa relies on readily inducible protection to combat desiccation and desiccation tolerance is controlled by multiple genes within the plant nucleus. Thirdly, the protective mechanisms of desiccation tolerance utilized by X. viscosa appear to involve signal perception genes and modulating gene expression of appropriate genes encoding protective molecules including antioxidants, molecular chaperones, compatible solutes, proteins of translation and degradation machinery, proteins with miscellaneous functions and novel protein factors. Lastly, proteins are crucial to desiccation tolerance allowing X. viscosa to possess a unique stress tolerance with versatile and coordinated actions to provide protection for its cellular structures during desiccation and in the dried state. To our best knowledge this is the first study to provide insight into the nuclear (organellar) proteome of a desiccation tolerant plant.
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    The anti-fungal and anti-oxidant properties of polyphenols extracted from the resurrection plant, Myrothamnus flabellifolia
    (2008) Shibambo, Segopotjo Linah; Lindsey, George G; Brandt, Wolf F
    In this study the effects of M. flabellifolia polyphenols on growth of S. cerevisiae yeast strains was investigated. This study showed that M flabellifolia polyphenols inhibited growth of both the wild type and the Δhsp 12 yeast strains largely by binding protein in the growth medium. A decreased specific growth rate, reduced maximum biomass, and prolonged lag phase were observed for both strains.
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    Antiretroviral drugs differentially modulate glucocorticoid activity via the glucocorticoid receptor in vitro
    (2019) Kuipa, Michael; Hapgood, Janet; Moliki, Mosoko; Maritz, Michelle
    Concurrent use of anti-retroviral drugs (ARVs) and progestin-based hormonal contraceptives is widespread. During times of stress and during glucocorticoid (GC) therapy, intracellular ARVs are in the presence of high concentrations of GCs, which regulate all aspects of immunity and inflammation via the glucocorticoid receptor (GR). However, the reciprocal modulation of ARV and steroid intracellular functions is relatively unexplored. In this study, the effects of the ARVs tenofovir disoproxil fumarate (TDF), dapivirine (DPV), and maraviroc (MVC) on activation of the GR and GR-regulated mRNA expression were investigated, in the absence and presence of select GR ligands. The effects of TDF and DPV on GR protein levels and phosphorylation were also determined. The inhibitory activity of these ARVs on HIV-1 infection in the presence of the progestins medroxyprogesterone acetate (MPA) and levonorgestrel (LNG), and a GR agonist, dexamethasone (DEX) was also assessed. This study shows that (0.01 nM-10 µM) TDF, DPV and MVC do not transactivate reporter gene expression via the unliganded GR exogenously expressed in the steroid receptor-deficient U2OS human osteosarcoma cell line, or alter the reporter gene transcriptional activity of (100 nM) MPA or LNG via the GR in these cells. However, (1 µM) TDF and DPV modulate the reporter gene transcriptional efficacy of (0.01 nM-10 µM) DEX via the GR. In the U2OS cell line model, (1 µM) TDF, but not DPV significantly decreased (1µM and 10µM) DEX-induced mRNA expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. TDF also appeared to decrease (1 µM) cortisol (CORT)- and MPA-induced GILZ mRNA expression. This may be mediated by the apparent increase in (100 nM and 1µM) DEXinduced phosphorylation at Serine 226 on the GR, observed in the presence of (1µM) TDF in this study. DPV and TDF (at 1µM) did not significantly alter GR protein levels, or cell-viability in the absence and presence of (100 nM) DEX, CORT or MPA in U2OS cells. However, (1 µM) DPV and TDF alone, significantly altered cell viability in peripheral blood mononuclear cells (PBMCs). In PBMCs, (1 µM) TDF, MVC and DPV alone altered basal GILZ mRNA expression and had variable, donor-specific effects on interleukin (IL)-6, IL-8, and interferon (IFN)-γ gene expression. In PBMCs from some of the nine donors tested, these ARVs had proinflammatory effects which may undermine their efficacy at preventing HIV-1 acquisition in pre-exposure prophylaxis products. Moreover, the ARVs proinflammatory effects may negatively impact HIV-1 disease progression and increase the risk of non-AIDS mortality in individuals using the ARVs therapeutically. Neither (1 µM) DPV, TDF nor MVC significantly altered the effects of (100 nM) DEX on the immunomodulatory genes assessed in PBMCs. DEX, MPA and LNG (at 100 nM) did not affect the anti-HIV-1 activity of the ARVs (at 1 µM) in PBMCs from the majority of the three donors tested in this study. Taken together, the results show that ARVs can modulate GR activity in an ARV-, steroid-, gene- and cell-specific manner, while the steroids investigated did not modulate ARV anti-HIV-1 activity.
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    The arabidopsis cyclic nucleotide interactome
    (BioMed Central, 2016-05-11) Donaldson, Lara; Meier, Stuart; Gehring, Christoph
    Background: Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotidedependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods: An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results: A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions: We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.
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    Architecture and assembly of maize streak virus: insights from 3D electron microscopy
    (2014) Dent,Kyle Clayton; Varsani, Arvind; Sewell, Trevor
    Maize streak virus (MSV), circular single stranded DNA (ssDNA) virus (~2.7kb), is the causative agent of Maize streak disease, and is a devastating pathogen that causes severe crop losses to subsistence farmers in sub-Saharan Africa. MSV is transmitted by the leafhopper Cicadulina mbila, and is the type member of the Mastrevirus genus (family Geminiviridae). MSV shares a unique twinned icosahedral ("geminate") virion architecture (22 x 38 nm) with all other family members. Geminate particles consist of 110 coat protein (CP) subunits that assemble onto a circular single-stranded DNA (ssDNA) genome. Each T= I unit is an incomplete icosahedron assembled from 55 CPs. The structures of MSV and African cassava mosaic virus (ACMV, genus Begomovirus) have been studied by electron cryo- microscopy (cryo-EM) previously. While these investigations revealed some details about the geminate architecture, the interactions of capsid components have not yet been adequately modelled. The two incomplete icosahedral "heads" of the geminate particle are offset from one another and apparently make distinct CP:CP contacts at this region of the virion. Information regarding the nature of quasi- equivalent CP conformers or the sets of amino acid residues that mediate these interactions has not been forthcoming. Since the experimental results of these previous studies are not available in a public database, we were motivated to revisit the structure of MSV in order to obtain a 3D experimental density that might aid pseudo-atomic modelling. The MSV CP:ssDNA interaction has also been shown to be crucial for systemic movement through the host. Hence, quasi-atomic modelling may inform development of antiviral strategies which aim to interfere with virion assembly. MSV virions were isolated from the leaves of maize plants infected by agro-inoculation and visualized in both heavy metal stain and vitreous ice after they had been adsorbed to a thin-layer of continuous carbon to prevent virion aggregation. Virus preparations consisted of distinct CP assemblies consisting of multiples of the incomplete T=I icosahedral unit. Monopartite (icosahedral), bipartite (geminate), tripartite, and higher assemblies were observed suggesting the MSV CP is not only multifunctional but also structurally versatile being able to package ssDNA of variable sizes. Low-dose images were recorded on film, and 3D reconstruction of both monopartite and bipartite capsid species carried out using standard single-particle image processing methodology. The resolution of the bipartite reconstructions was 26 A for the negative-stain dataset, and 23 A for cryo-EM dataset, while the resolution of the monopartite reconstruction was estimated to be ~15 A. Comparative modelling of the MSV CP was undertaken using the pentamer (CPs) of Satellite tobacco necrosis virus (STNV) as a structural template. Correlation-based fitting of icosahedral and geminate atomic models that varied in geometric arrangement of MSV CPs allowed the geometric parameters of the bipartite capsid to be determined. Fitting ofMSV CPs into the EM densities informed our understanding of interfaces which allow the CP to self-associate, and showed that CPs is in fact displaced within the icosahedral geometry of the heads by a 10° rotation about the 5-fold axes of symmetry in comparison to STNV; hence, while quaternary structure of the pentameric capsomer is conserved between these viruses, the quaternary interactions between capsomers of the T=I unit has diverged considerably. This study shows that the offset between the geminate heads of the MSV virion is ~-11°, and that this geometry appears to arise owing to a distinct set of CP:CP interfaces which occur across the equator between two quasi-icosahedral heads and involve regions that would interact to form the CPs: CPs interfaces within each of the heads (across 2-fold and 3-fold symmetry axes). Notably this offset differs from that reported for ACMV, which has a reported offset of 20°. Additionally, the resolution afforded by the icosahedral monopartite reconstruction provided the first structural evidence to suggest that the calcium ion binding site of the STNV CPs (located on the CS axis) is likely to be conserved in MSV. This result suggests that in common with other plant viruses, depletion of calcium ions may be required for genome egress in a newly infected host cell. This study highlights the importance of future high-resolution studies of this unique virion morphology by both X-ray crystallography and cryo-EM.
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    ASMT gene polymorphisms are associated with Autism Spectrum Disorder (ASD) symptom severity in a South African population
    (2016) De Waal, Margaretha; O'Ryan, Colleen; Roden, Laura
    Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by behavioural and social impairments. ASD shows evidence of a genetic aetiology, with a large body of research linking ASD to polymorphisms in several different genes and gene families, including those involved in circadian rhythm generation and melatonin biosynthesis. Sleep disorders are highly comorbid with ASD in both children and adults, and range from sleep onset delay, phase shift and sleep disruption. These parasomnias can have a significant impact on the quality of life for persons with ASD and their families, and sleep deprivation can feed into the behavioural deficits in ASD. Melatonin supplementation is often prescribed to assist in alleviating the above mentioned sleep dysfunction. Melatonin is a hormone in the circadian clock system, and is a biochemical signal for darkness to synchronise peripheral cells to the master oscillator. Clinical trials reported that melatonin supplementation at night assists in sleep initiation. However both the mode of action of supplemental melatonin, as well as whether melatonin deficiency is common in ASD, remains unclear. Furthermore, any research on ASD is often hamstrung by the heterogeneous nature of the disorder, necessitating clear phenotyping. This study examines single nucleotide polymorphisms (SNPs) in the gene acetylserotonin methyl transferase (ASMT), which encodes an enzyme in melatonin biosynthesis, in a South African ASD cohort (n=28) and controls (n=6). All participants completed and Autism Diagnostic Observation Schedule-2 assessment that allowed partitioning of the ASD individuals into ASD endophenotypes, to reduce phenotyping heterogeneity. This study found SNPs previously associated with ASD in the promoter and intronic region. Additionally, this study found novel SNPs, and a SNP in a putative transcription factor binding site not previously associated with ASD. The associations found between SNPs and ASD endophenotypes, together with the positions of the SNPs, suggest a potential link between ASMT polymorphisms and ASD symptom severity. Further research, using language assessment tools as well as quantitative measures of melatonin and sleep disruption, may establish the role of melatonin in language impairment in ASD.