Research Articles (Biotechnology)

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Development of infectious transcripts and genome manipulation of black queen-cell virus of honey bees
(Microbiology Society, 2002) Benjeddou, Mongi; Leat, Neil; Allsopp, Mike
The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5«-proximal ORF encoding a putative replicase protein and a 3«-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)–PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered.
Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana
(Elsevier, 2003) Ludidi, Ndiko; Gehring, Chris
Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3 ,5 -cyclic monophos- phate (cGMP) from guanosine 5 -triphosphate (GTP). While many cGMP-mediated processes in plants have been reported, no plant molecule with GC activity has been identified. When the Arabidopsis thaliana genome is queried with GC sequences from cyanobacteria, lower and higher eukaryotes no unassigned proteins with sig- nificant similarity are found. However, a motif search of the A. thaliana genome based on conserved and func- tionally assigned amino acids in the catalytic center of annotated GCs returns one candidate that also contains the adjacent glycine-rich domain typical for GCs.
Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form
(Wiley, 2007) Thuku, Ndoria R.; Weber, Brandon W.; Varsani, Arvind
Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1–327.
A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye
(Future Science Group, 2008) Meyer, Mervin; Essack, Magbubah; Kanyanda, Stonard
We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V–based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.
Alteration of Bax-to-Bcl-2 ratio modulates the anticancer activity of methanolic extract of Commelina benghalensis (Commelinaceae) in Jurkat T cells
(Academic Journals, 2008) Mbazima, Vusi G.; Mokgotho, Matlou P.; February, Faghri; Rees, Jasper G.; Mampuru, Leseilane J.
Stem extracts of Commelina benghalensis (Linn.), although not extensively documented, are frequently used in traditional medicine for the treatment of ailments such as skin malformations and outgrowths. Accordingly, the study was aimed to investigate possible molecular mechanisms that are associated with the potential anti-carcinogenic property of this agrofield weed. Jurkat T cells were exposed to different concentrations (0-600 ug/ml) of the crude methanolic extract of C. benghalensis to evaluate their growth inhibitory and apoptosis inducing effects. The extract elicited a dose- and time-dependent inhibition of cell proliferation, followed by a concomitant decrease in cell viability. The observed cytotoxicity was linked to the induction of apoptosis as determined by morphological and biochemical features known to be associated with the advent of apoptosis. Real time quantitative RT-PCR and Western blot analyses of Bax, Bcl-2 and p53 exhibited aberrant expression profiles of these genes under various treatment conditions. Taken together, the data suggest that the crude methanolic extract of C. benghalensis contains bioactive compounds that may be beneficial in the treatment of malignant growths, and that this apparent antineoplastic activity is a consequence of dysregulated expression of apoptosis-responsive genes. These observations could provide a credible scientific justification upon which the ethnopharmacological utilisation of C. benghalensis is founded.
Identification of a novel alkaliphilic esterase active at low temperatures by screening a metagenomic library from Antarctic desert soil
(American Society for Microbiology, 2009) Heath, Caroline; Hu, Xiao Ping; Cary, Craig
The cold deserts of the McMurdo Dry Valleys, South Victoria Land, Eastern Antarctica, are widely acknowledged as having the harshest soil environments on Earth (6, 8, 26). Despite the apparent hostility of the environment, we and others have reported both unexpectedly high biomass (9) and phylogenetic diversity (1, 19, 24, 29) in Antarctic soils. The presence of numerous novel taxa suggests that these soils might prove to be valuable sources of genetic material for mining novel industrial enzymes active at low temperatures (9, 23). Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester compounds. Their applications in industry cover a broad spectrum, including as detergent additives, in food processing, in environmental bioremediation, and in biomass and plant waste degradation for the production of useful organocompounds (3, 16).
Ozone and nitric oxide induce cGMP-dependent and independent transcription of defence genes in tobacco
(Wiley, 2009) Pasqualini, Stefania; Meier, Stuart; Gehring, Chris
Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) and the role of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses. Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3, but is essential and sufficient for the induction of some O3-dependent pathways.
The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is coexpressed with pathogen defense related genes
(Public Library of Science, 2010) Meier, Stuart; Ruzvidzo, Oziniel; Morse, Monique
Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.
Extremophiles in biofuel synthesis
(Taylor and Francis Group, 2010) Barnard, Desire; Casanueva, Ana; Tuffin, Marla
The current global energy situation has demonstrated an urgent need for the development of alternative fuel sourcesto the continually diminishing fossil fuel reserves. Much research to address this issue focuses on the developmentof financially viable technologies for the production of biofuels. The current market for biofuels, defined as fuelproducts obtained from organic substrates, is dominated by bioethanol, biodiesel, biobutanol and biogas, relying onthe use of substrates such as sugars, starch and oil crops, agricultural and animal wastes, and lignocellulosic biomass.This conversion from biomass to biofuel through microbial catalysis has gained much momentum as biotechnologyhas evolved to its current status. Extremophiles are a robust group of organisms producing stable enzymes, which areoften capable of tolerating changes in environmental conditions such as pH and temperature. The potentialapplication of such organisms and their enzymes in biotechnology is enormous, and a particular application is inbiofuel production. In this review an overview of the different biofuels is given, covering those already producedcommercially as well as those under development. The past and present trends in biofuel production are discussed,and future prospects for the industry are highlighted. The focus is on the current and future application ofextremophilic organisms and enzymes in technologies to develop and improve the biotechnological production ofbiofuels.
Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells
(Public Library of Science, 2011) Sharathchandra, Ramaschandra G.; Stander, Charmaine; Ndimba, Bongani
This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first.
Unique aliphatic amidase from a psychrotrophic and haloalkaliphilic nesterenkonia isolate
(American Society for Microbiology, 2011) Nel, A. J. M.; Tuffin, I. M.; Sewell, B. T.
Nesterenkonia strain AN1 was isolated from a screening program for nitrile- and amide-hydrolyzing microorganisms in Antarctic desert soil samples. Strain AN1 showed significant 16S rRNA sequence identity to known members of the genus. Like known Nesterenkonia species, strain AN1 was obligately alkaliphilic (optimum environmental pH, 9 to 10) and halotolerant (optimum environmental Na+ content, 0 to 15% [wt/vol]) but was also shown to be an obligate psychrophile with optimum growth at approximately 21°C.
Mapping and characterisation of the sorghum cell suspension culture secretome
(Academic Journals, 2011) Ngara, Rudo; Ndimba, Bongani K.
Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two-dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well-resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and ?-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against ?-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop.
Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings
(Elsevier, 2012) Ngara, Rudo; Ndimba, Roya; Borch-Jensen, Jonas; Jensen, Ole Nørregaard; Ndimba, Bongani K.
Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghumvariety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.
Nodulation, leaf harvesting intensity and interval of the black-eyed bean (BEB) (Vigna unguiculata L. Walpers) cowpea type at the University of Zimbabwe, Harare, Zimbabwe
(Academic Journals, 2012) Matikiti, A.; Chikwambi, Z.; Nyakanda, C.; Mashingaidze, A.B.
A glasshouse pot experiment was carried out at the University of Zimbabwe, Crop Science Department in 2006 to 2007 rainy season to determine the effect of leaf harvesting intensity and interval on cowpea biological nitrogen fixation through nodulation. The experiment was designed as a three factor factorial. The first factor was leaf harvesting frequency with two levels: leaves harvested weekly or fortnightly. The second factor was leaf harvesting intensity with three levels: harvesting one leaf, two leaves and three leaves per branch. The third factor was soil with two levels: Mutoko sand and UZ red clay soil. The experiment was arranged in a Randomized Complete Block Design (RCBD) with three replications. There was no significant difference (p>0.05) on the number of nodules formed at different intensities and frequency at 6, 9 and 12 weeks after crop emergence. Significant difference (p<0.05) in nodule weight was observed as it decreased with increase in leaf harvesting intensity. Harvesting weekly produced lower nodule weight (0.31 g) than harvesting fortnightly (1.08 g). Mutoko sand soil produced higher nodule weight (0.69 g) than UZ clay soil (0.43 g). Leaf yield increased with increasing leaf harvest intensity, harvesting weekly produced higher yield (10.43 g) than harvesting fortnightly (3.22 g) and Mutoko soil had lower leaf yield than UZ clay soil. There was a significant (p<0.05) difference in seed yield, highest yield was obtained when no leaves were harvested and lowest yield when three leaves were harvested. Soil type effect also significantly (p<0.05) reduced harvestable seed weight from 1.00 g on sand soil to 4.12 g on clay soil. Similar trends were observed for aboveground and total biomass yield. In this study, leaf harvesting intensity and interval has been observed to affect nodulation, grain and biomass yield of glasshouse grown Black-eyed bean (BEB) cowpea. The extent to which these effects were felt also depends on the characteristics of the soil.
Modulation of antioxidant enzyme activities and metabolites ratios by nitric oxide in short-term salt stressed soybean root nodules
(Elsevier, 2013) Egbichi, I.; Keyster, Marshall; Jacobs, Alex; Klein, Ashwil; Ludidi, Ndiko
Several abiotic factors cause molecular damage to plants either directly or through the accumulation of reactive oxygen species such as hydrogen peroxide (H2O2). We investigated if application of nitric oxide (NO) donor 2,2′- (hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) could reduce the toxic effect resulting from short-term salt stress. Salt treatment (150 mM NaCl) alone and in combination with 10 μM DETA/NO or 10 μM DETA were given to matured soybean root nodules for 24 h. Salt stress resulted in high H2O2 level and lipid peroxidation while application of DETA/NO effectively reduced H2O2 level and prevented lipid peroxidation in the soybean root nodules. NO treatment increased the activities of ascorbate peroxidase and dehydroascorbate reductase under salt stress. Whereas short-term salt stress reduced AsA/DHAsA and GSH/GSSG ratios, application of the NO donor resulted in an increase of the reduced form of the antioxidant metabolites thus increasing the AsA/DHAsA and GSH/GSSG ratios. Our data suggests a protective role of NO against salt stress.
Nitric oxide (NO) regulates the expression of single-domain cystatins in glycine max (soybean)
(Southern Cross Publishing, 2013) Keyster, Marshall; Adams, Ruqaiyah; Klein, Ashwil; Ludidi, Ndiko
Plant cystatins inhibit cysteine proteases and are important in regulating plant development and plant responses to biotic and abiotic stress. Furthermore, nitric oxide plays a signaling role in regulating plant responses to developmental processes, biotic and abiotic stress. With the aim of determining if nitric oxide is involved in the regulation of the expression of single-domain cystatins, we have identified single-domain cystatin genes in soybean (Glycine max cv. PAN626) on the basis of sequence homology to a nitric oxide-inducible cystatin (AtCYS1, At5g12140) from Arabidopsis thaliana. Analysis of the expression of the four cystatin genes revealed that transcript levels of these cystatins are altered by exogenously applied nitric oxide and a nitric oxide synthase inhibitor. Altered expression of these cystatins by nitric oxide and the nitric oxide synthase inhibitor implies that changes in cellular nitric oxide content, which have previously been shown to occur during development and/or biotic and abiotic stress, influence soybean physiological processes that are regulated by cysteine proteases. Recombinant protein expression of one of the cystatins (as a glutathione-S-transferase fusion protein) showed that it has inhibitory activity against the model cysteine protease papain but not the model serine protease trypsin and that it inhibits caspase-like activity in soybean nodule extracts. This serves as evidence that these four plant cystatins are functional cysteine protease inhibitors because of their high degree of primary sequence identity. It also indicates that the single-domain cystatins regulate caspase-like activity, which is known to participate in plant responses to biotic and abiotic stress. We thus conclude that nitric oxide and nitric oxide synthase-like activity regulate the expression of these cystatins, thus influencing soybean caspase-like activity. We also propose a role for this nitric oxide-mediated regulation of cystatin gene expression in the mediation of developmental processes and responses to abiotic stress in soybean.
Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds
(Ivyspring, 2013) Ndimba, Bongani K.; Ibraheem, Omodele
Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of ‘intelligent’ metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.
One-pot synthesis and characterization of InP/ZnSe semiconductor nanocrystals
(Elsevier, 2013) Mushonga, Paul; Onani, Martin O.; Madiehe, Abram M.; Meyer, Mervin
We report on the one-pot synthesis of InP/ZnSe quantum dots (QDs) using a precursor-based colloidal synthesis in a noncoordinating solvent environment. The structural and optical studies were carried out on the as-prepared InPQDs. The quantum yield of the nanocrystals was recorded as 6% and a 1.4 times reduction in the ratio of trap-related emission to band edge emission was observed on ZnSe passivation of the InPQDs.
Association analysis of two single-nucleotide polymorphisms of the RELN gene with autism in the South African population
(Mary Ann Liebert, Inc., 2013) Sharma, Jyoti Rajan; Arieff, Zainunisha; Gameeldien, Hajirah; Davids, Muneera; Kaur, Mandeep; van der Merwe, Lize
BACKGROUND: Autism (MIM209850) is a neurodevelopmental disorder characterized by a triad of impairments, namely impairment in social interaction, impaired communication skills, and restrictive and repetitive behavior. A number of family and twin studies have demonstrated that genetic factors play a pivotal role in the etiology of autistic disorder. Various reports of reduced levels of reelin protein in the brain and plasma in autistic patients highlighted the role of the reelin gene (RELN) in autism. There is no such published study on the South African (SA) population. AIMS: The aim of the present study was to find the genetic association of intronic rs736707 and exonic rs362691 (single-nucleotide polymorphisms [SNPs] of the RELN gene) with autism in a SA population. METHODS: Genomic DNA was isolated from cheek cell swabs from autistic (136) as well as control (208) subjects. The TaqMan® Real-Time polymerase chain reaction and genotyping assay was utilized to determine the genotypes. RESULTS: A significant association of SNP rs736707, but not for SNP rs362691, with autism in the SA population is observed. CONCLUSION: There might be a possible role of RELN in autism, especially for SA populations. The present study represents the first report on genetic association studies on the RELN gene in the SA population.
Capacity to control oxidative stress-induced caspase-like activity determines the level of tolerance to salt stress in two contrasting maize genotypes
(Springer, 2013) Keyster, Marshall; Klein, Ashwil; Du Plessis, Morné; Jacobs, Alex; Kappo, Abidemi; Kocsy, Gábor; Galiba, Gábor; Ludidi, Ndiko
The response of two maize (Zea mays L.) genotypes, named GR (salt-tolerant) and SK (salt-sensitive), to salt stress (150 mM NaCl) was investigated under controlled environmental growth conditions. Genotype SK experienced more oxidative damage than the GR genotype when subjected to salt stress, which corresponded to higher O2- production rate and H2O2 content in the SK genotype than the GR genotype. Induction of caspase-like activity in response to salt stress was stronger in the SK genotype than in the GR genotype. On the other hand, induction of antioxidant enzyme activity to scavenge O2- and H2O2 in response to salt stress was weaker in the SK genotype than in the GR genotype. Consequently, the higher level of oxidative damage in the SK genotype in response to salt stress was manifested as more extensive cell death and biomass reduction in the SK genotype than it was in the GR genotype. Our results suggest that a direct relationship exists between salt stress-induced oxidative damage and cell death-inducing caspase-like activity, with tolerance to the salt stress being controlled by the efficiency of the plantantioxidant enzymes in limiting salt stress-induced oxidative damage and thus limiting cell death-inducing caspase-like activity.

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