Department of Biochemistry

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Synthesis of biogenic gold nanoparticles from terminalia mantaly extracts and the evaluation of their in vitro cytotoxic effects in cancer cells
(MDPI, 202-09-12) Sharma, Jyoti R.; Sibuyi, Nicole Remaliah Samantha; Majoumouo, Michele Stella
Scientists have demonstrated the potential of plant materials as 'green' reducing and stabilizing agents for the synthesis of gold nanoparticles (AuNPs) and opened new ecofriendly horizons to develop effective and less harmful treatment strategies. The current study demonstrated the use of Terminalia mantaly (TM) extracts to synthesize AuNPs with enhanced cytotoxic effects. The TM-AuNPs were synthesized at 25 and 70 °C using water (WTM) and methanolic (MTM) extracts of the leaf, root and stem/bark parts of the plant. The TM-AuNPs were characterized using UV-visible spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy, energy dispersive X-ray (EDX), selection area electron diffraction (SAED) and Fourier transform infrared (FTIR) spectroscopy. Majority of the TM-AuNPs were spherical with a mean diameter between 22.5 and 43 nm and were also crystalline in nature. The cytotoxic effects of TM-AuNPs were investigated in cancer (Caco-2, MCF-7 and HepG2) and non-cancer (KMST-6) cell lines using the MTT assay. While the plant extracts showed some cytotoxicity towards the cancer cells, some of the TM-AuNPs were even more toxic to the cells. The IC50 values (concentrations of the AuNPs that inhibited 50% cell growth) as low as 0.18 µg/mL were found for TM-AuNPs synthesized using the root extract of the plant. Moreover, some of the TM-AuNPs demonstrated selective toxicity towards specific cancer cell types. The study demonstrates the potential of TM extracts to produce AuNPs and describe the optimal conditions for AuNPs using TM extracts. The toxicity of some the TM-AuNPs can possibly be explored in the future as an antitumor treatment.
"Genetic variation between two subspecies of reedfrogs in the genus Hyperolius (Anura: Hyperoliidae)"
(University of the Western Cape, 1993) Hess, Anthony Jacobus; Hendricks, Denver
H. m. broadleyi and H. m. verrucosus are not only different in terms of colour pattern, but distinct genetic differences were detected in restriction site maps of their ribosomal DNA. A sequence divergence value of 13,8% was found between the subspecies. This value exceeds the range recorded between separate species of the genus Rana, ie. 2,2 % between Rana pustulosa and Rana tarahumarae, and 10, 1 % between Rana pustulosa and Rana pipiens. The value of 13,8% between the subspecies is also in the same range as that found between was detected between samples from three different localities within the distribution range of H. m. verrucosus. The genetic data associated with the different colour patterns, suggests that H. m. broadleyi and H. m. verrucosus can be regarded as distinct species. However, a similar study should be performed to examine the genetic status of the subspecies forming the gradient along the east coast of southern Africa. Although the effect of concerted evolution (as discussed in Chapter 2) allows for small sample sizes it would be useful to examine a large number of individuals especially from the overlap zones to determine the extent of genetic heterozygosity and to determine if similar genetic differences (found between H. m. broadleyi and H. m. verrucosus) exist between the rest of the subspecies. The current study has shown that genetic evaluation of all the southern African H. marmoratus subspecies could have a positive impact on the taxonomy of this group of frogs which is still unresolved. This study has identified at least two species within the H. marmoratus complex and it is possible that more species exist within the group. Because of morphological homogeneity it will be difficult to use morphological characters, but more than one molecular technique can be utilized to verify results obtained with one technique.
Genetic variation between two subspecies of Reedfrogs in the genus Hyperolius (Anura: Hyperoliidae)
(University of the Western Cape, 1993) Hess, Anthony Jacobus; Hendricks, Denver
The frog genus Hyperolius is the largest of 19 genera in the Hyperoliidae (Channing, 1939). The members of the genus display considerable morphological homogeneity with a diversity of dorsal colour patterns. The genus is endemic to Africa and is distributed throughout the wetlands south of the Sahara excluding the western plateau slopes and South African plateau. They are found in close proximity to water, on reeds, sedges and also on water-lily leaves (Iambiris, 1989). The South African forms are commonly referred to as reedfrogs (Passmore and Camrthers, 1979). The members of the genus bury themselves in the ground and under leaves during the dry season and emerge after the first rains (Iambiris, 1989). The eggs are laid in water, attached to waterplants, but certain species deposit eggs above the water level or between water-lily leaves (Lambiris, 1989). Characters that disqinguish the genus from other African genera are as follows: The pupil is horizontal to round; Vomerine teeth are absent; The fingers and toes are webbed; The oral disc is ventrally situated (Passmore and Camrthers, 1979).
Genetic variation of rana fuscigula in Southern Africa
(University of the Western Cape, 1994) Arieff, Zainunisha; Channing, Alan
Naturalists have long been engaged in describing and explaining diversity in the biological world. The discovery of the molecular basis of inheritance has led to rapid increase in the use of biological macromolecules in these investigations. Scientists now routinely investigate the DNA of a range of organisms. The elationships between taxa and the phylogeny of groups is determined by examining the differences and similarities between them. These differences are then appropriately analyzed. lt is important to understand the natural variation within a group, before the differences between groups can be established. This study aims to determine the molecular differences between individuals at the extreme edges of the distribution of a species. This will serve as a molecular baseline, from which other studies can proceed. The experimental species is the trog Rana fuscigula, which has a range restricted to southern Namibia and South Africa. lt was thus possible to collect material from the edge of the distribution assuming that maximum genetic difference would be found between individuals at the edge of the range
The adenomatous polyposis coli (APC) and p53 gene status in South African oesophageal cancer patients
(University of the Western Cape, 1996) Skelton, Michelle; Hendricks, Denver; Smith, Arlene
Oesophageal cancer is the most conmon cause of cancer-related deaths in black males in South Africa. In an effort to understand the molecular nature of oesophageal carcinogenesis in South Africa, two tumour suppressor genes, Adenomatous Polyposis Coli (APC) and p53, were examined in normal and tumour tissue obtained from 33 oesophageal cancer patients. Several studies have shown that alterations of the APC and p53 genes are associated with the development of cancer. Allelic loss at the APC gene locus was examined using two polymorphic markers within the coding region of the APC gene. Single stranded conformation polymorphism, heteroduplex and DNA sequencing analyses were used to detect mutations in the mutation susceptible regions of the APC and p53 genes. The "Mutation Cluster Region" (MCR) in exon 15 of the APC gene was examined. Only exons 5 and 6 in the "hot spot" region of the p53 gene were examined. An allelic loss of 21% (4 of 19 informative cases) and an informativity of 59Yo (19 of 32) of the APC gene was demonstrated in patients analysed. No somatic mutations were detected in the MCR in exon 15 of the APC gene. Three putative mutations were detected in the p53 gene using SSCP and HD analysis, two of which were confirmed by DNA sequencing. Analysis of one patient revealed a TCA to TGA base substitution at codon 183 in exon 5 of the p53 gene, resulting in a stop codon at that position. An eleven base pair deletion in exon 6 of the p5 3 gene was detected in another patient. This deletion caused a frame shift mutation and culminated in a premature stop codon 13 codons downstream. Overall, a mutation frequency of 8% (2 of 25 patients analysed) was detected for the p53 gene with exons 7 and 8 still pending further study. These results suggest that the APC gene may not be involved in oesophageal cancer in South Africa and further studies are necessary in order to examine the role of thep53 gene in this disease in South Africa.
Aberrations in the Retinoblastoma susceptibility gene in tumours from South Africa oesophageal cancer patients
(University of the Western Cape, 1996) Gamieldien, Junaid; Hendricks, D.T; Smith, A; Parker, M.I
Little is known about the genetic events occurring in oesophageal cancer and very few studies have been undertaken to analyse oesophageal tumours from South African patients in this regard. Inactivation of numerous tumour suppressor genes, including the Rb gene, has been implicated in oesophageal tumourigenesis in different populations. This study had two objectives. The first was to develop a procedure for the simultaneous extraction of DNA and RNA from small (ca. 25mg) oesophageal biopsy samples. The procedure developed here has proven to be rapid, cost effective and consistently produced excellent yields of high-quality DNA and RNA. It has to be determined, however, whether long-term storage affects the integrity of the isolated RNA. The second and primary objective of this study was to determine whether the Rb gene is involved in oesophageal tumourigenesis in South African patients. Loss of Heterozygosity analysis using a VNTR marker in intron 20 and a microsatellite marker in intron 4 of the Rb gene revealed that Rb-allelic loss had occurred n 50% of the thirty-three patients analysed. Furthermore, microsatellite instability was demonstrated at the intron 4 marker in 15% of the patients analysed. Mutation screening of exons 17 and 21 of the Rb gene, frequently mutated in oesophageal tumours from Chinese patients, in twenty samples using the mutation screening techniques of SSCP and heteroduplex analysis, followed by DNA sequencing of putative positives, revealed no positive mutations. However, the high percentage of allelic loss found suggests that the /lb gene is inactivated in the progression of South African oesophageal tumours. Furthermore, the microsatellite instability suggests that defective DNA repair may also play a role in oesophageal tumourigenesis.
Identification and partial characterisation of a putative novel plant stress response gene from Arabidopsis thaliana.
(University of the Western Cape, 2000) Ludidi, Ndiko Ndomelele; Gehring, C. A.
Exposure of plants to stress results in the expression of plant genes whose products play a role in defence responses against the stress. Stress stimuli to which plants may be exposed are wounding, drought, salinity, excessive light intensity, heat stress, pathogens, biotic and abiotic factors that lead to the accumulation of H2O2, salicylic acid and plant hormones e.g absiscic acid. This study describes the identification and partial characterisation of a putative novel gene from Arabidopsis thaliana, named the DIINN gene. The sequence of the predicted protein encoded by the DWNN gene shows similarity to a gene isolated from Chinese hamster ovary cells that were resistant to chemically induced programmed cell death. Since programmed cell death is one of the processes involved in plant defence responses to stress, it is hypothesised that the DWNN gene may also play a role in plant programmed cell death. The protein product encoded by the DWNN gene, DWNN, shows homology to proteins from diverse species and phyla. Plants transformed to overexpress DWNN show severely stunted growth and abnormal developmental patterns while plants in which DWNN has been knocked out show an accelerated growth rate. Analysis of the expression pattem of the DLI/NN gene using the GUS gene reporter system suggests that the DWNN gene is expressed in secondary lignification during xylogenesis and in wounded plant tissue. Both xylogenesis and wounding are processes known to involve progralnmed cell death and the regulation of protein turnover.
Novel genomic approaches for the identification of virulence genes and drug targets in pathogenic bacteria
(University of the Western Cape, 2001) Gamieldien, Junaid; Hide, Winston; Faculty of Science
While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence –associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.
Isolation and Partial Characterization Serine Protease Inhibitors from Triticum aestivum cv' Witwol
(University of the Western Cape, 2001) Morse, Monique; Bradley, Graeme; Klaasen, Jeremy
Plant diseases and plant pests are major constraints to plant growth and development, resulting in severe crop losses annually. Plant pathogens can be a variety of things, living and non-living. Non-living pathogens exist as physical conditions that plants are exposed to- climatic conditions can cause damage to plants and there are various agricultural practices that can also be harmful to the plant. Living pathogens are called parasitic or infectious diseases and are extremely contagious and can spread from plant to plant very quickly. Insects, nematodes, mites and higher animals can be considered as pathogens, as are slime mold, bacteria, fungi and parasitic higher plants. Viruses and viroids are also considered pathogens (URL ). Fungal diseases, in particular, severely limit the production of major crops, as do insect crop pests. Effective control of pathogens has led to widespread use of chemical fungicides and insecticides, but with potentially deleterious environmental and human health consequences associated with this practice, strategies to utilize natural host plant resistance mechanisms for disease control are being pursued (Yun e/ a1.,1997). The agrochemical industry has been actively looking for less damaging ways to control insect pests, and has introduced a number of more environmentally friendly pesticides. In addition, alternative strategies for pathogen and pest control have been pursued, such as biological control, and the use of plant varieties with inherent resistance
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.
Expression and purification of the novel protein domain DWNN
(University of the Western Cape, 2002) Lutya, Portia Thandokazi; Pugh, David J.R.; Faculty of Science
Proteins play an important role in cells, as the morphology, function and activities of the cell depend on the proteins they express. The key to understanding how different proteins function lies in an understanding of the molecular structure. The overall aim of this thesis was the determination of the structure of DWNN domains. This thesis described the preparation of samples of human DWNN suitable for structural analysis by nuclear magnetic resonance spectroscopy (NMR), as well as NMR analysis.
The effect of kinetin and salt stress on pnp-a expression in erucastrum strigosum and arabidopsis thaliana
(University of the Western Cape, 2002) Makgoke, Gile Dineo; Gehring, C. A.
In agriculture crop productivity is greatly affected by stresses such as salinity, drought, temperature and honnonal changes of crop plants and responses to these stresses. Studies have shown that a natriuretic peptide based regulatory system responsible for water and ion homeostasis in animals has a hctcrologous equivalent in plants. Plant natriuretic peptide immunoreactants (irPNPs) have been reported to be involved in K+, Na+ and er ions fluxes in plants. Previously, an Arabidopsis thaliana transcript (AtPNP-A) encoding an irPNP (AtPNP-A) has been identified and isolated (Ludidi et al., 2002). The AtPNP-A a novel protein and part of its physiological role is presented here.
Low detection of exon skipping in mouse genes orthologous to human genes on chromosome 22
(University of the Western Cape, 2002) Chern, Tzu-Ming; Hide, Winston; Faculty of Science
Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes. Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves.
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.
The development and application of informatics-based systems for the analysis of the human transcriptome
(University of the Western Cape, 2003) Kelso, Janet; Hide, Winston; Faculty of Science
Despite the fact that the sequence of the human genome is now complete it has become clear that the elucidation of the transcriptome is more complicated than previously expected. There is mounting evidence for unexpected and previously underestimated phenomena such as alternative splicing in the transcriptome. As a result, the identification of novel transcripts arising from the genome continues. Furthermore, as the volume of transcript data grows it is becoming increasingly difficult to integrate expression information which is from different sources, is stored in disparate locations, and is described using differing terminologies. Determining the function of translated transcripts also remains a complex task. Information about the expression profile – the location and timing of transcript expression – provides evidence that can be used in understanding the role of the expressed transcript in the organ or tissue under study, or in developmental pathways or disease phenotype observed. In this dissertation I present novel computational approaches with direct biological applications to two distinct but increasingly important areas of research in gene expression research. The first addresses detection and characterisation of alternatively spliced transcripts. The second is the construction of an hierarchical controlled vocabulary for gene expression data and the annotation of expression libraries with controlled terms from the hierarchies. In the final chapter the biological questions that can be approached, and the discoveries that can be made using these systems are illustrated with a view to demonstrating how the application of informatics can both enable and accelerate biological insight into the human transcriptome.
Bioactivity guided fractionation of Sutherlandia frutescens extracts for the induction of apoptosis
(University of the Western Cape, 2003) Chinkwo, C.A; Gehring, C.A
Sutherlandia frutescens popularly known as "cancer bush" or "kankerbos" is indigenous to South Africa and claimed by traditional healers to have wide therapeutic potential, most specifically against cancer. To verify these traditional claims we used apoptosis-based bioassays, organic solvent extraction, TLC (thin layer chromatography) and HPLC (high perforrnance liquid chromatography) to evaluate extracts from Sutherlandia frutescens microphylla from different geographical populations and from selected S. frutescezs subspecies. The data demonstrate that a specific Sutherlandia frutescens extract has the ability to induce apoptosis in cultured cells. This investigation has suggested that the induction of apoptosis by the extract shows some specificity for transformed cultured cells. In addition, biological activity was traced by chemical fractionation of the crude extract to the chloroform and ethyl acetate fractions. Extracts obtained from S. frutescens microphylla from different geographical regions and subspecies were compared, there was variation in apoptotic activity between the extracts. This suggests that the apoptotic activity and hence possible anti-cancer activity of this plant depends on external environmental factors. In summary, the presented findings are supportive of the claims made by traditional healers that S. frutescens has anti-cancer activity. However we have found that apoptotic activity was not present in all the plants, even of the same species, and hence further investigations are required to identify which factors cause certain plants to have greater apoptotic activity than others. Furthermore the extract analyzed in this study must be further characterized to identify compounds with anti-cancer activities.
Physical and genetical investigation of the Xp11.3 region on the short arm of the human X-chromosome
(University of the Western Cape, 2004) Wittwer, Pia Ethena; Gehring, Chris; Dept. of Biochemistry; Faculty of Science
The pattern of inactivation in the DXS8237E-UBE1-PCTK1 region is of particular interest, since the mechanisms of X chromosome inactivation and the escape from inactivation are, as yet, not fully understood. The inactivation status of the DXS8237E and PCTKl gene differ: the first undergoes normal inactivation and the second escapes this process. The status of the UBEl gene has been controversial, although it is widely excepted that it does escape X chromosome inactivation. Physical mapping of the region employing YACs and subsequently P ACs has been undertaken, but was restricted in scope by the high frequency of rearrangements occurring. DNA sequences between DXS8237E, UBE1, PCTKl and the distal gene, UHX1, have been investigated with regard to LINEI elements, which are thought to playa role in X-inactivation. The results obtained strongly suggest a link between LINE1 elements and X chromosome inactivation. Sequence analysis results also contributed to the understanding of difficulties with restriction mapping of the region. Further, this work includes the first reported establishment of the UBEl exonintron boundaries. Additionally, genomic sequence analysis showed that only 46kb separate DXS8237E from UHX1, which confirms that this region is extremely gene rich.
Optimizing the ion source for polarized protons
(University of the Western Cape, 2005) Johnson, Samantha; Celliers, P.; Faculty of Science
Beams of polarized protons play an important part in the study of the spin dependence of the nuclear force by measuring the analyzing power in nuclear reactions. The source at iThemba LABS produces a beam of polarized protons that is pre-accelerated by an injector cyclotron (SPC2) to a energy of 8 MeV before acceleration by the main separated-sector cyclotron to 200 MeV for physics research. The polarized ion source is one of the two external ion sources of SPC2. Inside the ion source hydrogen molecules are dissociated into atoms in the dissociator and cooled to a temperature of approximately 30 K in the nozzle. The atoms are polarized by a pair of sextupole magnets and the nucleus is polarized by RF transitions between hyperfine levels in hydrogen atoms. The atoms are then ionized by electrons in the ionizer. The source has various sensitive devices, which influence beam intensity and polarization. Nitrogen gas is used to prevent recombination of atoms after dissociation. The amount of nitrogen and the temperature at which it is used plays a very important role in optimizing the beam current. The number of electrons released in the ionizer is influenced by the size and shape of the filament. Optimization of the source will ensure that beams of better quality (a better current and stability) are produced.
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.

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