Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
Genetic and biological characterisation of a novel South African Plutella xylostella granulovirus (PlxyGV) isolate
- Authors: Abdulkadir, Fatima
- Date: 2014
- Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
- Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
- Full Text:
- Date Issued: 2014
Isolation, propagation and rapid molecular detection of the Kalahari truffle, a mycorrhizal fungus occurring in South Africa
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2007
Structural bioinformatics analysis of the Hsp40 and Hsp70 molecular chaperones from humans
- Authors: Adeyemi, Samson Adebowale
- Date: 2014
- Subjects: Structural bioinformatics , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Biomolecules
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4171 , http://hdl.handle.net/10962/d1020962
- Description: HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.
- Full Text:
- Date Issued: 2014
Biochemical characterisation of Pfj2, a Plasmodium falciparum heat shock protein 40 chaperone potentially involved in protein quality control in the endoplasmic reticulum
- Authors: Afolayan, Omolola Folasade
- Date: 2013
- Subjects: Plasmodium falciparum Endoplasmic reticulum Heat shock proteins Malaria , Mosquito-borne infectious disease
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3883 , http://hdl.handle.net/10962/d1001617
- Description: Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease
- Full Text:
- Date Issued: 2013
Stress-inducible protein 1: a bioinformatic analysis of the human, mouse and yeast STI1 gene structure
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
The role of parallel computing in bioinformatics
- Authors: Akhurst, Timothy John
- Date: 2005
- Subjects: Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3986 , http://hdl.handle.net/10962/d1004045 , Bioinformatics , Parallel programming (Computer science) , LINDA (Computer system) , Java (Computer program language) , Parallel processing (Electronic computers) , Genomics -- Data processing
- Description: The need to intelligibly capture, manage and analyse the ever-increasing amount of publicly available genomic data is one of the challenges facing bioinformaticians today. Such analyses are in fact impractical using uniprocessor machines, which has led to an increasing reliance on clusters of commodity-priced computers. An existing network of cheap, commodity PCs was utilised as a single computational resource for parallel computing. The performance of the cluster was investigated using a whole genome-scanning program written in the Java programming language. The TSpaces framework, based on the Linda parallel programming model, was used to parallelise the application. Maximum speedup was achieved at between 30 and 50 processors, depending on the size of the genome being scanned. Together with this, the associated significant reductions in wall-clock time suggest that both parallel computing and Java have a significant role to play in the field of bioinformatics.
- Full Text:
- Date Issued: 2005
Bacterial degradation of ixodicide amitraz
- Authors: Allcock, Errol Ralph
- Date: 1978
- Subjects: Ticks -- Control , Pesticides -- Biodegradation , Acaricides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4081 , http://hdl.handle.net/10962/d1007473 , Ticks -- Control , Pesticides -- Biodegradation , Acaricides
- Description: The control of ticks on cattle has long been a matter of prime importance to stock owners over most of the intensive natural grazing areas in the Southern Hemisphere. The only practical method of dealing with the cattle tick problem in the short term is by treating the infected bovine host with ixodicides i. e. by chemical control. This can be achieved by either plunging the cattle into a dip tank containing aqueous suspensions or emulsions of the ixodicide or by spraying them with dip suspensions in a spray race.
- Full Text:
- Date Issued: 1978
Serotonin-melatonin interactions in acetaminophen and N,N-dimethylformamide toxicity
- Authors: Anoopkumar-Dukie, Shailendra
- Date: 2000
- Subjects: Serotonin , Acetaminophen , Melatonin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3898 , http://hdl.handle.net/10962/d1003957 , Serotonin , Acetaminophen , Melatonin
- Description: Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.
- Full Text:
- Date Issued: 2000
An investigation into the antioxidative potential and regulatory aspects of liver tryptophan 2,3-dioxygenase by tryptophan and related analogues
- Authors: Antunes, Ana Paula Martins
- Date: 1998
- Subjects: Tryptophan -- Physiological effect , Antioxidants , Liver
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4010 , http://hdl.handle.net/10962/d1004070 , Tryptophan -- Physiological effect , Antioxidants , Liver
- Description: The amino acid, tryptophan, obtained through dietary means, is metabolised by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase. All the enzymes have an effect on circulating tryptophan levels, especially TDO, since it is the major site of tryptophan catabolism in the liver and results in the production of kynurenine metabolites, viz. kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and quinolinic acid. Extrahepatically, IDO is responsible for the synthesis of the kynurenine metabolites. Tryptophan 2,3-dioxygenase and IDO activity is increased by hormones or substrates such as tryptophan, and inflammation, in the case of IDO. Tryptophan availability for serotonin (5-HT) synthesis by the enzyme tryptophan hydroxylase is primarily dependent on TDO activity. A study was attempted in order to ascertain whether any of the endogenous metabolites of the kynurenine and serotonergic pathways would be able to inhibit TDO activity. Results showed that although the kynurenines had no effect, the indoleamines, except for the indoleacetic acids, were able to reduce TDO activity. 6-Methoxy-2-benzoxazolinone (6-MBOA), a structural analogue to melatonin, was the most potent inhibitor with a reduction in activity of 55 % compared with the control. The pineal gland in the rat brain has been shown to have the highest IDO activity. With induction, the kynurenine metabolite concentrations of kynurenic acid and quinolinic acid are increased. The effects of both compounds were determined on the serotonergic pathway. Although kynurenic acid produced no significant effect, quinolinic acid significantly reduced N-acetylserotonin and melatonin synthesis at concentrations of lOJLM and 100 JLM respectively. Many authors have implicated oxygen derived species as causative agents in the important neurodegenerative disorders such as Parkinson's and Huntington's disease. Increased radical generation and lipid peroxidation have been suggested to be responsible for the toxic destruction of neurons, especially in the brain because of its high lipid content and oxygen demand. The brain is therefore vulnerable to oxidative attack. During inflammatory diseases, IDO is induced with a resultant increase in kynurenines. This study was also an attempt at determining the effect of kynurenines on lipid peroxidation. All metabolites of the kynurenine pathway were able to induce lipid peroxidation significantly. The antioxidative potential of various tryptophan analogues, viz. serotonin, melatonin and 6-methoxy-2-benzoxazolinone, was determined using quinolinic acid-induced lipid peroxidation. Serotonin, melatonin and 6-MBOA were able to significantly reduce quinolinic acid-induced lipid peroxidation.
- Full Text:
- Date Issued: 1998
Studies on the ecology and systematics of the diatoms (Bacillariophyta) from some South Africa rivers
- Authors: Archibald, Robert Eldred Mostert
- Date: 1969
- Subjects: Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4098 , http://hdl.handle.net/10962/d1009494 , Diatoms -- Ecology -- South Africa , Diatoms -- Classification , Aquatic ecology -- South Africa
- Description: This report contains the results of some ecological and systematic studies on the diatoms from the Vaal Dam catchment area in the Transvaal, and the Bloukrans River in the Eastern Cape Province. In Part 1 the effects of high concentrations of nitrogen were studied in relation to the composition of the diatom associations. Water samples from four stations on the Bloukrans River were analysed chemically at certain intervals during the months of April and August 1967. Diatom samples collected from these stations at the beginning and end of each of these sampling periods were subjected to a "Thomasson Analysis" to determine the relative densities of the various species in the diatom associations. A statistical analysis of the results reflected a poor but positive correlation between the two variables, i.e. high numbers of nitrogen heterotrophic Nitzschiae were correlated with high concentrations of nitrogen, while low numbers were correlated with low concentrations. Part 2 presents the results of the ecological studies on the diatom associations of the Vaal Dam Catchment Area. In this section the diatom associations from each sampling point or station were subjected to a "Thomasson Analysis" to determine the relative densities of the different species in the associations. Employing already known correlations between environment and association, the results of this analysis were discussed and the ecological conditions for each sampling station were assessed. The associations were similar in composition over the entire catchment area, and indicated on the whole water of good quality. Points of pollution were detected, but were generally localised and the effects of the pollution were soon removed. Only the Waterval River showed evidence of more constant pollution. The associations provided evidence for some seasonal variation in their composition. Finally in Part 3 the systematics and taxonomy of the diatoms in the Vaal Dam catchment area are discussed. References are made to the original and more recent descriptions of each species found in this study, and a list of synonyms is given wherever 'possible. Comments on the systematics, taxonomy and autecology of each species are given, and the distribution of the species in South Africa and the Vaal Dam catchment area is discussed. A number of species, varieties and forms have been recorded for the first time in South Africa. During the course of this study 20 species have been described as new to science; the descriptions of some have been published, while the descriptions of the others will be published formally in the future. All species described as new or having interesting features are illustrated in the plates.
- Full Text:
- Date Issued: 1969
Regulation of the indoleamines by sex steroids
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Date Issued: 1992
Bacterial degradation of the acaricide amitraz
- Authors: Baker, Penelope Bridget
- Date: 1976
- Subjects: Acaricides , Biodegradation , Gas chromatography , Bacteriology -- Cultures and culture media
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4099 , http://hdl.handle.net/10962/d1009498
- Description: This thesis describes dip tank field trials and laboratory investigations on the acaricide Amitraz. Amitraz is a triazapenta- diene compound which is relatively unstable in fouled dip washes. The field trials were conducted on the farm Sea View according to the "Total Replacement Method" and on the farm Sea Ways according to the "Lime Stabilization Method" of dipping. The results of these trials showed that Amitraz was stable in clean dip washes, and under conditions of high pH resulting from the addition of slaked lime to the dip wash. Using mixed bacterial populations optimum conditions for degradation of Amitraz in the laboratory were determined. Bacterial cultures degraded Amitraz most efficiently in media supplemented with yeast extract or with a high content of sterile cattle faeces. Amitraz concentrations were determined by gas chromatography. A culture. efficient at degrading Amitraz was enriched from a dip tank sludge inoculum. From this culture ten bacterial isolates were identified; nine of these were of the genus Pseudomonas and one was an Achromobacter sp. Experiments with both mixed and pure cultures demonstrated that bacterial degradation of Amitraz was by the process of co-metabolism. The existence of four degradation products was shown using thin layer chromatography. Tentative identification of two of the products was made.
- Full Text:
- Date Issued: 1976
Studies on the fermentation of molasses by Clostridium acetobutylicum
- Authors: Barber, Jennifer Mary
- Date: 1978
- Subjects: Molasses , Clostridium acetobutylicum , Fermentation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4084 , http://hdl.handle.net/10962/d1007611 , Molasses , Clostridium acetobutylicum , Fermentation
- Description: The bacterium Clostridium acetobutylicum produces acetone and n [subscript] - butanol from molasses in an industrial fermentation system. Although the bacterium has been cultured in liquid media it does not grow well on agar plates and requires high concentrations of hydrogen. Pretreatment of agar plates with bovine catalase improves growth on agar media. The bacteria produce an area of clearing (halo) on Potato agar plates due to butyric acid (the precursor of n [subscript]-butanol) and ß -amylase production. This characteristic will be used as a plate screening assay for the selection of high solvent producing mutants. A laboratory scale fermentation system was developed and detailed studies including pH, turbidity and cell morphology changes, and the details of solvent production were undertaken. The fermentation was optimized for mutant selection. The production of normal solvent yields by isolated clones is required for the mutant selection programme. Studies revealed that sporulation of the clones increased their solvent yield although solvent yields were still lower than normal. Efficient sporulation is therefore a prerequisite for clone fermentation. The origin of the phage infection during the factory outbreak was determined and resistant clones obtained. The presence of a bacteriocin-like toxin causing decreases in turbidity was identified during the final fermentation stage. The strain sensitivity, optimum conditions for stability as well as the kinetics of inactivation and lethality have been investigated. Preliminary characterization and purification studies indicate the proteinaceous nature of the toxin. , KMBT_363
- Full Text:
- Date Issued: 1978
Understanding of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Date Issued: 2020
Epidemiological and aetiological aspects of diarrhoeal disease in the Eastern Cape
- Authors: Baxter, Esther
- Date: 1993
- Subjects: Diarrhea -- South Africa , Intestines -- Diseases , Pathogenic microorganisms -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4045 , http://hdl.handle.net/10962/d1004106 , Diarrhea -- South Africa , Intestines -- Diseases , Pathogenic microorganisms -- South Africa
- Description: Diarrhoeal disease is a major cause of mortality in children in developing countries. It also remains a serious problem among all age groups throughout the world. Whereas studies to determine the epidemiological and aetiological factors of diarrhoeal disease have been reported for other parts of South Africa and the world, as yet no information is available for the Eastern Cape. Therefore this study was undertaken to determine the factors for this area. Enteropathogens were compared for the different ages in the various population groups and, where possible, seasonal and geographical differences were emphasised. A total of 7 278 faecal samples were examined by six laboratories in the Eastern Cape during the period November 1988 to October 1990. Data was recorded noting the age, sex and population group of the patients. The towns selected were Port Elizabeth, Uitenhage, Cradock, Grahamstown and their surrounding areas. The isolation rates for the pathogens studied in the various population groups were compared to those reported in similar studies in other countries. The seasonal incidences of the various selected pathogens were compared with those reported from elsewhere in South Africa. It was thought that the higher temperature of summer may influence the finding in the White population group, while rainfall would play a greater role for the Coloured and Black populations. The geographical distribution of the pathogens emphasised the difference in living conditions between the different population groups. For example a generally higher infestation rate of Helminths occurred in rural areas and in the groups living under poorer conditions. The low isolation rates for certain bacteria and the large percentage of samples from which no pathogens were isolated indicate the need for further research. However, the finding should be valuable for determining Public Health priorities and in the management of outbreaks of diarrhoeal disease.
- Full Text:
- Date Issued: 1993
Identification of SANCDB compounds against G2019S and I2020T variants of leucine-rich repeat Kinase 2 (LRRK2) for the development of drugs against Parkinson’s Disease
- Authors: Baye, Bertha Cinthia
- Date: 2020
- Subjects: Antiparkinsonian agents , Parkinson's disease -- Treatment , Protein kinases , Parkinson's disease -- Chemotherapy , Molecules -- Models
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/138764 , vital:37671
- Description: Parkinson’s disease is a type of movement disorder that occurs when nerve cells in the brain stop producing dopamine. It is the second neurodegenerative disease affecting 1-2% of people above the ages of 65 years old. There is a worldwide prevalence of 7 to 10 million affected people of all cultures and race. Studies have shown that mutation that causes Parkinson’s disease result in increased kinase activity. The c.6055 G > A in exon 41 is the most prevalent LRRK2 variation which causes a substitution of glycine to serine in G2019S in the highly activated loop of its MAP kinase domain. The LRRK2 G2019S variant is the most common genetic determinant of Parkinson’s disease identified to date. This work focused on building accurate 3D models of the LRRK2 kinase domain, that were used for large-scale in silico docking against South African natural compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). Molecular docking was performed to identify compounds that formed interactions with the active site of the protein and had the lowest binding energy scores. Molecular dynamics simulations showed different movements of the protein-ligand complexes and behavioural difference of the wildtype and the variants, all three structures proved to be compact. Network analysis was done to study residue interactions, contact maps, dynamic cross correlations, average BC and average L were used to study the residue interactions and general residue contribution to the functioning of the protein..
- Full Text:
- Date Issued: 2020
An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
Ericoid mycorrhizal fungi and potential for inoculation of commercial berry species (Vaccinium corymbosium L.)
- Authors: Bizabani, Christine
- Date: 2011
- Subjects: Ericaceae , Mycorrhizas , Fynbos
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4136 , http://hdl.handle.net/10962/d1016127
- Description: Ericaceous plants are the richest growth form of the fynbos vegetation of South Africa. The fynbos is characterized by highly leached acidic soils, low mineral nutrients and climatically it is a winter rainfall and dry summer region. Ericoid mycorrhizal fungi associate with Erica species enhancing their ability to access essential nutrients for survival under unfavourable growth conditions. The aim of this study was to select local Ericaceae plant species and to isolate, identify and characterize the ericoid endophytes and assess these isolates as potential inocula for commercial berry species. Two ericaceous plants Erica cerinthoides L. and Erica demmissa Klotzsch ex Benth. were identified from the Mountain Drive area of Grahamstown, Eastern Cape. Root staining was used to confirm the mycorrhizal status of both plants. Hyphal coils typical of ericoid association were observed within the epidermal cells of the hair roots under a light microscope. The endophytes were successfully isolated in pure culture on 2% malt extract agar (MEA) and modified Fontana medium. Cultural morphology and microscopy were used for initial identification. Two slow growing isolates were selected. These isolates were further subjected to molecular identification; extracted DNA was amplified using ITS1 and ITS4 fungal primers. The rDNA gene internal transcriber spacer (ITS) was then sequenced and analyzed by comparison to sequences in the GenBank. On the basis of percentage sequence identity Lachnum Retz. species and Cadophora Lagerb. & Melin species were identified as the ericoid endophytes of E. cerinthoides and E. demmissa respectively. The optimum growth parameters of the fungal isolates were determined in 2% MEA incubated at varying temperatures and pH. It was established that both species had optimum growth at 27⁰C and pH 5. The Ericaceae species are sometimes found in metal contaminated sites were ericoid fungi have been proved to alleviate toxicity of their host. The fungal isolates were grown in increasing concentration of Cu²⁺ and Zn²⁺ in 2% MEA. The growth of Lachnum species decreased with increasing Zn²⁺ ions above 2.7 mM while Cadophora species showed a change in morphology and also decreased in growth with increased ion concentration. However there were no significant differences recorded in the growth of Cadophora and Lachnum species on increasing Cu²⁺ concentration. Lachnum and Cadophora isolates were formulated into a semi solid inoculum and inoculated onto micropropagated Vaccinni corymbosum L. plantlets of 5 different varieties. Colonization was low for all varieties, Elliott and Brightwell varieties recorded the highest colonization of 35% and 31% respectively. Lachnum species infected roots showed potential ericoid structures while the Cadophora inoculated plantlets had hyphal coils within the cortical cells typical of ericoid mycorrhizas. Inoculation significantly enhanced the shoot growth of Brightwell and Elliott varieties. The Chandler variety inoculated with Lachnum species showed improved shoot dry weight. The Bluecrop and Elliott varieties inoculated with Cadophora and Lachnum accumulated more root biomass. All inoculated Bluecrop plantlets had an improved canopy growth index. Brightwell plantlets inoculated with Lachnum species also had an enhanced canopy growth index. The growth responses were variable within varieties and between varieties. Treatments with the Cadophora and Lachnum have shown potential in the promotion of growth of the Blueberry species. The findings indicate the need to conduct trials under conditions which simulate the commercial growth conditions so as explore the optimum potential of the isolates.
- Full Text:
- Date Issued: 2011
Cleavage of the precursor coat protein of black beetle virus strain w17 in rabbit reticulocyte lysate
- Authors: Blackhurst, Diane Mary
- Date: 1988
- Subjects: Beetles , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3880 , http://hdl.handle.net/10962/d1001614
- Description: Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure
- Full Text:
- Date Issued: 1988