OpenUCT :: Browsing by Department "Division of Chemical Pathology"

Browsing by Department "Division of Chemical Pathology"

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Open Access
A new approach to the serological classification of paracolons, utilising trichloracetic acid extraction of polysaccharide antigenic fractions, with observations on biochemical classification, pathogenicity and taxonomy and review of the literature
(1954) MacPherson, Colin Robertson
Open Access
A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles
(2001) Champeil, Philippe; Henao, Fernando; Lacapère, Jean-Jacques; McIntosh, David B
After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.
Open Access
Acute Rheumatic Fever and Rheumatic Heart Disease: Highlighting the Role of Group A Streptococcus in the Global Burden of Cardiovascular Disease
(2022-04-21) Auala, Tangeni; Zavale, Ben’Lauro Goncalves; Mbakwem, Amam Çhinyere; Mocumbi, Ana Olga
Group A Streptococcus (GAS) causes superficial and invasive infections and immune mediated post-infectious sequalae (including acute rheumatic fever/rheumatic heart disease). Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are important determinants of global cardiovascular morbidity and mortality. ARF is a multiorgan inflammatory disease that is triggered by GAS infection that activates the innate immune system. In susceptible hosts the response against GAS elicits autoimmune reactions targeting the heart, joints, brain, skin, and subcutaneous tissue. Repeated episodes of ARF—undetected, subclinical, or diagnosed—may progressively lead to RHD, unless prevented by periodic administration of penicillin. The recently modified Duckett Jones criteria with stratification by population risk remains relevant for the diagnosis of ARF and includes subclinical carditis detected by echocardiography as a major criterion. Chronic RHD is defined by valve regurgitation and/or stenosis that presents with complications such as arrhythmias, systemic embolism, infective endocarditis, pulmonary hypertension, heart failure, and death. RHD predominantly affects children, adolescents, and young adults in LMICs. National programs with compulsory notification of ARF/RHD are needed to highlight the role of GAS in the global burden of cardiovascular disease and to allow prioritisation of these diseases aimed at reducing health inequalities and to achieve universal health coverage.
Open Access
Antioxidant roles of uric acid and tyrosine in mammalian erythrocytes
(2003) Matshikiza, Maia Thandi; Harley, Eric
Open Access
Apolipoprotein E variants, plasma lipids, lipoproteins and dysApolipoprotein E variants, plasma lipids, lipoproteins and dysβLipoproteinaemia during pregnancy in Zimbabwean women.
(2005) Tanyanyiwa, Donald Moshen; Marais, A D
This study of pregnant women in Zimbabwe therefore set itself the following aims: To describe lipid and lipoproteins during and after pregnancy, To examine the prevalence of apoE variants, To evaluate dysβlipoproteinaemia in pregnancy, The correlation between dysβplipoproteinaemia and the apoE genotypes. This is the first study to systematically examine lipids and lipoproteins during pregnancy in black Africans.
Open Access
Asn 102 of the Gonadotropin-releasing Hormone Receptor Is a Critical Determinant of Potency for Agonists Containing C-terminal Glycinamide
(1996) Davidson, James S; McArdle, Craig A; Davies, Peter; Elario, Ricardo; Flanagan, Colleen A; Millar, Robert P
We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.
Open Access
Aspects of purine and pyrimidine metabolism
(1989) Black, Duncan Arthur; Harley, Eric H
In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.
Open Access
ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p
(1998) Decottignies, Anabelle; Grant, Althea M; Nichols, J Wylie; de Wet, Heidi; McIntosh, David B; Goffeau, André
The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.
Open Access
Autocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons
(1994) Pithey, Anne Louise; Millar, Robert P; Dutlow, Clive
The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release.
Open Access
A bacteriological and pathological investigation of tuberculosis meningitis in the Western Province of the Cape of Good Hope.
(1952) Coetzee, Jack Nicol
Although there have always been sporadic reports of spontaneous cures in tuberculous meningitis, up to a few years ago this diagnosis almost invariably meant death within a few weeks or months. The therapeutic use of Streptomycin in this disease has created new hope for these patients and has stimulated tremendous interest in all aspects of the disease.
Open Access
The biochemical analysis of southern African rhinoceros populations
(1993) O'Ryan, Colleen; Harley, Eric H
The drastic decline in the numbers of the five extant species of rhinoceroses world-wide, mainly as a result of poaching, have placed these species in imminent danger of extinction. This emphasizes the need to understand the relationships among the different species of rhinoceros. The advances in molecular biology have allowed the application of DNA-based genetic techniques to address a number of aspects of rhinoceros biology which have both academic interest and practical value to conservation management. There are four aspects to this study: Firstly, restriction endonuclease maps of mitochondrial DNA were constructed to estimate the time of divergence of Diceros bicornis (black rhinoceros) and Ceratotherium simum (white rhinoceros) from their common ancestor. Secondly, a population genetic study of the relationships among four subspecies of D. bicornis. Thirdly, the application of DNA fingerprinting to examine the intra- and inter-population relatedness in D. bicornis populations. Fourthly, a practical application of PCR to identify the origin of an unknown sample of DNA.
Open Access
Biochemical and genetic properties of HPRT Cape Town
(1987) Galloon, Terry; Harley, Eric H
An unusual partial HPRT deficient mutant, HPRT Cape Town was observed to have a low activity in erythrocyte lysates at high concentrations of the purine substrates, hypoxanthine and guanine. This substrate inhibition was not observed with the substrate PPRP. The low activity was not associated with changes in the Km or Vmax for any of the substrates (Steyn and Harley, 1984). The kinetics of the proband's enzyme was studied in lymphoblast extracts. The characteristic substrate inhibition was observed which showed that this phenomenon was not confined to erythrocytes but was a more generalized phenomenon. This result implies that the decreased HPRT activity observed in the proband is due to substrate inhibition by the purine bases. The HPRT enzyme is coded for by a gene which is located on the X chromosome (Pai et al., 1980). The proband's daughter was therefore studied in order to determine the cause of the mutation. It was not known whether the substrate inhibition was the result of a mutation in the gene coding for the enzyme, a mutation which results in altered post-translational modification or the absence or alteration of factors influencing normal HPRT kinetics. The daughter's transformed lymphoblasts exhibited growth patterns in selective media that resembled those of her father. The daughter's enzyme prepared from lymphoblast extracts exhibited the characteristic substrate inhibition. These results suggest that this cell line results from the selection of a clone or clones which have suppressed the function of the X chromosome carrying the maternal and presumably normal HPRT allele. The daughter's enzyme prepared from erythrocyte lysates exhibited intermediate enzyme activity between that of the proband and a normal control. This result suggests that the daughter is an obligate heterozygote and that the defect is due to a mutation in the HPRT gene itself. The defect was studied at the gene level. No difference was observed in the banding patterns of the proband's DNA and control DNA which were digested with various restriction enzymes and hybridized to ³²p-labelled HPRT cDNA. The size of the HPRT mRNA of the proband was the same as the control. These results imply that there is no major gene alteration; this is expected since the proband only has a partial deficiency of the enzyme. The HPRT cDNA was subcloned into a riboprobe vector, pGEM-3. The T7 promoter was used to transcribe antisense RNA strands which were then hybridized to the proband's RNA and control RNA. No difference was observed in the size of the protected fragment. This result does not exclude the possibility of a point mutation as the cause of the defect in HPRT Cape Town.
Open Access
The biochemical and molecular basis of Hypoxanthine-guanine phosphoribosyltransferase deficiency
(1996) Marinaki, Anthony Marin; Harley, Eric H
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyses the first step in purine salvage. A complete deficiency of the enzyme results in the devastating neurological symptoms of the Lesch-Nyhan syndrome. The Lesch-Nyhan syndrome is characterised by purine overproduction leading to, hyperuricemia and gout and a central nervous system disorder characterised by severe, spasticity, choreoathetosis, mental retardation and compulsive self-mutilatory behaviour, A partial deficiency of the enzyme results in purine overproduction, gout and occasionally, mild neurological symptoms. Patients are spared the compulsive self-mutilation of the Lesch-Nyhan syndrome. The major part of the thesis consists of the characterisation of the molecular defects in nine patients with the Lesch-Nyhan syndrome. The polymerase chain reaction was used to amplify reverse transcribed HPRT mRNA. The coding region of the amplified HPRT cDNA was either directly sequenced, or cloned and sequenced. All the mutations characterised were insertion or deletion events which resulted in premature termination of the predicted protein. Three patients were found to have a deletion of exon 7, two patients had single base insertions, while two patients appeared to have a complete deletion of the HPRT gene. An insertion in one patient was the result of a mutation within. intron 6 which created a new splice donor site. The new splice donor site in concert with a cryptic splice acceptor resulted in the creation of a new exon. A deletion of exons 2, 3 and 4 in another patient was found to lead to the alternative splicing of exon 5. These unusual splice junction mutations provided in viva support for the exon definition model of pre-mRNA splicing.
Open Access
Biochemical aspects of the idiopathic respiratory distress syndrome of the newborn
(1969) Hardie, Gwendoline; Kench, J E
This study was undertaken primarily to investigate the plasma protein system in infants with IRDS, as compared with healthy premature infants, as it had previously been reported that the plasma protein concentration in affected infants was abnormally low. It was attempted further to establish biochemical and/or immunological criteria for diagnosis of the disease and to discover reasons for the low IgG concentrations and raised α-fetoprotein concentrations found in the sera of these infants. Maternal serum proteins were also studied during pregnancy and at and after delivery of the infant. Interrelationships between α-fetoprotein, Human Growth Hormone and other proteins, in immunochemical systems were investigated. In summary, the main conclusions reached were as follows: (i) The total serum-protein concentration in affected infants is much reduced, as compared with healthy premature infants of the same gestational age. (ii) In IRDS infants, the relative and absolute concentration, of IgG is extremely low, whereas concentrations of other immune globulins, as far as could be determined, are within normal limits. (iii) Mothers of affected infants have significantly lower concentrations both of serum IgG and of IgM, than mothers of healthy premature infants. These changes in the serum-proteins are present throughout pregnancy. By six weeks post-partum, the IgG level has returned to normal, but the IgM level remains low. Concentrations of IgA and total serum-protein are normal at all times. (iv) Examination of oedema fluid, urine, faeces and amniotic fluid for γ-globulin content, has excluded the possibility that IgG is being lost from the circulation by these routes. (v) IRDS infants have, in their serum, agglutinins of the IgM type directed against the intact maternal IgG molecule. Similar agglutinins are present in a minority of healthy premature infants. Both IRDS and healthy infants have agglutinins against IgG fragments, in approximately 50% of cases. Agglutinin titres against these are similar in the two groups, but the incidence of agglutinins against Bence Jones protein type Lis raised in IRDS. (vi) Affected infants have an elevated serum concentration of α-fetoprotein, which disappears from the serum during the: first week of post-natal life. (vii) The majority of pregnant women examined have been observed to have serum agglutinins directed against α-fetoprotein. These cross-react with albumin prepared from sera of healthy adult males α-fetoprotein has been found in the serum of many pregnant women, especially during the second trimester. (viii) Immunological interrelationships between α-fetoprotein human serum albumin, Human Growth Hormone and human IgG have been demonstrated. (ix) Infants suffering from Rh-isoimmunization exhibit a serum- protein pattern similar to that seen in IRDS. Biochemical and immunological criteria for the diagnosis of IRDS have thus been established. The data to be presented indicate the presence of an immunological factor in the aetiology of the disease.
Open Access
Biochemical characterization of the nucleic acids of some human and animal viruses
(1982) Mew, Ronald Terence
In Part I, the isolation and partial characterization of human polyomaviruses from a number of renal transplant patients is described. These isolates proved refractory to cell culture propagation by the methods used, and were thus extracted directly from large volumes of patient's urine. This approach has the advantage that the virus cannot undergo any possible genomic modification, as tends to occur during adaptation to cell culture. Human polyomavirus DNA is very susceptible to mutation during cell passage. Four isolates from different patients yielded sufficient DNA for limited restriction endonuclease characterization. Surprisingly, all four gave the same patterns with EcoRI, BamHI and HindIII. Two isolates that were also digested with PstI gave an identical pattern. These patterns are similar to, but distinct from, other strains of the human polyomavirus BK that have been described. Our isolates had a similar-sized genome to BK, but only 3 HindIII sites compared with 4 in the prototype, and 2 PstI sites compared with only 1 in the prototype. The quantity of DNA obtained directly from urine was usually very limited. In order to produce adequate DNA for complete analysis, viral DNA was recombined with· a bacterial vector (pBR322) and cloned into Escherichia coli strains HB101 and C600. Initially, the well-studied strain BK(MM) was successfully cloned. This clone was used to prepare radioactively-labelled DNA probes for the detection of BK-specific sequences in urine isolates and in subsequent recombinants with patient material. Such cloned material is easier to prepare in bulk than DNA from virus passaged in cell culture. Early attempts to clone DNA from clinical isolates failed, but BK-specific DNA from a patient (P.R.) has recently been cloned successfully. These clones are presently being used to investigate the differences in sequence between our isolates and the known strains of BK. It is hoped that this will shed light on the mechanisms of gene expression of these potentially oncogenic viruses. In Part II, the genomes of four rotaviruses were studied. "Simian agent 11" (SAll) and "offal agent" (OA) were cell culture-adapted strains, whereas "epizootic diarrhoea of infant mice virus" (EDIM) and "infantile gastroenteritis virus" (IGV) were isolated from stool specimens. Experiments were performed to confirm the double-stranded RNA (dsRNA) nature of the SAll genome. It ran at a characteristic density of l.595g/ml in caesium sulphate density gradients, and was resistant to DNase and RNase at high ionic strengths, but susceptible to RNase at low ionic strength. At the start of the project few or no polyacrylamide gel pictures of the nucleic acids of these viruses had been published, although it was known they resembled reovirus in consisting of segmented double-stranded RNA. Such pictures were obtained, and molecular weight estimations made by comparison with dsRNA markers of known MW from a cytoplasmic polyhedrosis virus (CPV) from Heliothis armigera (Harley, Rubenstein, Losman and Lutton, 1977. Virology 76: 210-216). The difficulties in obtaining precise MW values for rotavirus genome segments are discussed. All four genomes consist of 11 dsRNA segments. The pattern of bands produced by PAGE is very similar, and high-resolution gels are required to detect the small mobility differences between some segments. Gel systems were developed to improve on the resolution obtained in co-electrophoresis experiments. During attempts to culture SAll and OA viruses in cell culture, it was observed that treatment of the cells and/or virus with versene-trypsin solution during infection gave a marked increase in virus yield. While this effect was being investigated, reports appeared on the potentiating effect of trypsin on the cell culture of previously refractory rotaviruses. We confirmed that trypsin, when present in the culture medium, greatly increased the yield of progeny SAll virus.
Open Access
The biochemical systematics of the Southern African Felidae
(1992) Mda, Nomusa Y; Harley, Eric H
The classification of the family Felidae (cats) is problematical due to the conservative nature of their morphology. Some workers classify the family into as many as 20 genera (Ewer, 1973) while others divide it into three genera (Walker et al., 1964). Such studies have largely been based on morphological and behavioural characters. Recently, molecular studies, namely, protein albumin immunological distances (Collier and O'Brien, 1985) and protein electrophoresis (Randi and Ragni, 1991) have been used to try and resolve the problems underlying this family. To complement the previous studies, in the present study we use mitochondrial (mt) DNA to construct a· phylogeny of eight members of the southern African Felidae namely, African wild cat, Felis lybica; domestic cat, Felis catus; caracal, Caracal caracal; European wild cat, Felis sylvestris; leopard, Panthera pardus; lion, Panthera leo; and cheetah, Acynonyx jubatus. Mitochondrial DNA (mt DNA) was utilized instead of nuclear DNA since it accumulates point mutations at a rate which is 5 to 10 times as fast as the nuclear DNA and is therefore particularly useful for studying more closely related organisms between sub-species, species and genera. Its apparent potential to be used as a tool for constructing genealogical trees and time scales makes it a method of choice in evolutionary studies. We used the restriction mapping approach to generate data for phylogenetic analysis. Restriction mapping was utilized since it gives good resolution at the species and genus level and evolutionary estimates derived from this method are considered more accurate than those obtained by methods such as the restriction fragment size comparison. We have also attempted to develop the methodology for sequencing part of the cytochrome b region of mt DNA following polymerase chain reaction (PCR) amplification. Both cladistic and distance approaches were used for phylogenetic construction. This study will be both of academic value and may have relevance to practical conservation management since these molecular approaches help to identify or confirm specific status especially with respect to the relationship between the domestic cat and the African and the European wild cats. Furthermore, such approaches can be used at the intraspecific level to address problems in biogeography and population genetics. Our results are in concordance with the previously determined morphological studies and albumin immunological distance studies. The restriction maps for the African wild cat and the domestic cat are identical, emphasizing their close relationship and the African origin of the domestic cat. The European wild cat showed a slight variation with the African wild cat or the domestic cat with four different restriction sites and a sequence divergence of 0.9. This suggests that the common ancestral mt DNA of these cats existed about 450 000 years ago. The lion and the leopard are monophyletic in both cladistic and distance approaches. The precise placement of caracal has yet to be resolved but it is deeply rooted in the phylogenetic analysis which would be more consistent with a separate generic status of the latter species rather than its inclusion within either Felis or Panthera. The distance analyses are consistent with the placement of the cheetah as the most distantly related species amongst the eight Felid species examined.
Open Access
Brain distribution and release of Cholecystokinin octapeptide
(1980) Hudson, Anne Mary; Millar, Robert P
In this thesis the in vitro release of immunoreactive CCK₈ (iCCK₈) from rat central nervous system preparations and the regulation of this release have been studied. Rat brain was dissected into the following regions; hypothalamus, cerebral cortex, striatum and thalamus, according to the method of Brownstein, Arimura, Sato et. al. (1975). CCK₈ was found to be distributed throughout these regions (range of 9 - 300 pmoles), with the highest concentration in cortex (300 pmol). In addition, low levels (range 9 - 30 pmoles) of CCK₈ were found in spinal cord, brain stem and cerebellum, in agreement with other workers. Immunohistochemical techniques have demonstrated CCK-like immunoreactivity in nerve cell bodies and fibres throughout brain, particularly in the cortex. Subcellular fractionation of rat brain was used to study the subcellular localisation of CCK. Tissue was homogenised to shear off nerve terminals (synaptosomes) which were purified and used to study the release of the peptide from hypothalamic and extrahypothalamic nerve endings.
Open Access
Characterisation of the reaction of 1,4-phenylenebismaleimide with Ca²⁺-ATPase and elucidation of the intramolecular crosslink site
(1997) Seekoe, Tshepo W; McIntosh, David B
The SR Ca²⁺-ATPase is an ATP driven pump that removes calcium from the sarcoplasm and myofibrils to allow muscle relaxation. The sulfhydryl crosslinker, 1,4- phenylenebismaleimide, reacts with Ca²⁺-ATPase (110 kD) to form a species with an apparent molecular weight of 125 kD, as well as dimers and high order oligomers, on SDS-P AGE. During the course of this study we have optimised and characterised the reaction of 1,4-phenylenebismaleimide with SR Ca²⁺-ATPase to produce the 125 kD species that is reminiscent of an E 125 species formed by intramolecular crosslink with glutaraldehyde. The glutaraldehyde crosslink involves the active site Lys 492 and Arg 678, in a zero distance link that overlaps with the ATP binding pocket, since it can be inhibited by nucleotides. It has been previously shown that the putative intramolecular crosslink with 1,4-phenylenebismaleimide is also sensitive to nucleotide binding. We show that the formation of the putative intramolecular crosslink of SR vesicles ( approximately 20 % of ATPase) with 1,4-phenylenebismaleimide is optimum at alkaline pH with micromolar concentrations of the crosslinker. The formation of ATPase dimers and high order oligomers, which were prominent in the reaction with SR vesicles, were eliminated by solubilising in Triton X-100. Under these conditions and in the presence of calcium, two intramolecular crosslinks are formed as seen in the formation of 125 and 130 kD species. The former seems to be in proximity of the y-phosphate and the latter in the β-phosphate region of the ATP binding site according to nucleotide protection studies. In the presence of detergent (Triton X-100) and absence of calcium, only the 125 kD species is formed and requires stabilisation by thapsigargin, a sesquiterpene lactone that binds the transmembrane α-helices. These conditions yield up to 60 % intramolecularly crosslinked ATPase. Trypsin digestion altered the apparent molecular weight of the 125 kD species to 135 kD, suggesting, in accordance with the results of glutaraldehyde crosslink, that the putative intramolecular crosslink 1s between tryptic fragments A and B. [¹⁴C]1,4-phenylenebismaleimide was synthesised to further characterise the reaction and to elucidate crosslinked amino acid residue following protein digestion, radioactive peptide purification, and sequencing. From filtration studies it was evident that a number of sulfhydryl residues were derivatized in both SR vesicles and solubilised Ca²⁺-ATPase. The results suggests that there is very fast reacting set of sulfhydryl groups, which could comprise of sulthydryls from Ca²⁺-ATPase and/or a minor contaminant protein as previous studies have indicated. Only this fast set was reduced by nucleotide binding. In Triton X-100, the total reactive residues increased two-fold and the biphasic nature of the curve showed that the intramolecular crosslink possibly involves a fast reacting sulfhydryl residue and a slow reacting one. Derivatization with [¹⁴C]1,4-phenylenebismaleimide followed by digestion and HPLC analysis revealed radio labelled peaks. Purification and sequencing of the adducts identified 8 reactive cysteines, namely Cys 12, Cys 344, Cys 364, Cys 471, Cys 498, Cys 636, Cys 670 and Cys 674. The cysteines involved in the putative intramolecular crosslink could not be identified but it is proposed that either Cys 471 or Cys 498 crosslink with Cys 670 or Cys 674.
Open Access
Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production
(1994) Shires, Karen Lesley; Harley, Eric H
A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection).
Open Access
Clinical and biochemical studies on the gluthathione S-transferases
(1982) Sherman, Morris; Kirsch, Ralph E
The glutathione S-transferases (ligandins) are a ubiquitous system of xenobiotic metabolising enzymes. In the rat liver they comprise up to 10% of soluble hepatic protein. Studies in the rat suggested that ligandin was an accurate and sensitive marker of hepatocellular necrosis. and of renal tubular necrosis. The first part of this thesis examines the release of ligandin from liver and kidney in human liver and renal disease in an attempt to determine whether the measurement of ligandin is clinically useful. Ligandin was purified from human liver cytosol using a combination of anion exchange chromatography and gel filtration. The purified protein had similar physicochemical characteristics to ligandin purified by others. The protein was used to raise a monospecific antibody. Ligandin was iodinated by the Chloramine-T method. which yielded a labelled protein of high specific activity. A sensitive and specific radioimmunoassay for human ligandin was developed which had a low intra- and interassay variation. The assay was applied to the study of human liver disease. In acute hepatitis ligandin is released from the liver into serum early in the illness. High serum ligandin levels are seen in the first week of acute hepatitis. The rapid return to normal suggests that ligandin may provide an early indication of recovery. In chronic hepatitis ligandin levels correlated significantly with histological severity of disease. whereas SGOT showed no such correlation. Ligandin may be a better index of severity of disease and for treatment than SGOT. Ligandin was released from the kidney in severe renal ischaemia and in acute tubular necrosis, but was not a reliable predictor or indicator of acute tubular necrosis. Part two examines the distribution of GSH-T activity in organs and in hepatocellular carcinoma. Ligandin was shown to be immunologically similar in all tissues studied. Isoelectric focusing of cytosol separated the three groups of GSH-T activity. Considerable variety in the distribution and activity of GSH-T's was shown in different organs from a single donor, and in the same organs from different donors. Anionic transferase activity was shown to contribute a significant proportion of activity in organs other than the liver. and to be the major source of activity in ovary and lung. In hepatocellular carcinoma cationic GSH-T activity was present in amounts varying from near normal to absent. The anionic and neutral GSH-T's were present in amounts similar to that seen in normal liver. Immunohistochemical studies using a peroxidase-antiperoxidase method showed a rough correlation between tumour differentiation and the amount of ligandin in the tumour.