OpenUCT :: Browsing by Author "Harley, Eric H"

Browsing by Author "Harley, Eric H"

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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
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
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
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
Kinetic and metabolic studies in HPRT deficiency
(1983) Steyn, Lafras Marais; Harley, Eric H
The patient (T.K.), was first diagnosed as having a partial hypoxanthine-guanine phosphoribosyltransferase deficiency in 1978 when he was 20 years old. At presentation, he complained of a colicky loin-pain which radiated into his groin, and that he had had dark urine for a month. He was shown to have haematuria and urate crystalluria, and had a serum urate of 0.8mmol/l (reference range 0.12-0.5mmol/l). The diagnosis was confirmed by demonstrating a haemolysate hypoxanthine-guanine phosphoribosyltransferase activity of 550μU/mg Hb (reference range 1680-2480μU/mg Hb). Studies to determine whether the low hypoxanthine-guanine phosphoribosyltransferase activity was caused by an altered Kₘ of the enzyme for one of its substrates, showed that there was substrate inhibition of the enzyme activity by hypoxanthine. This thesis examines the patient and the variant HPRT at three levels. Firstly, a detailed and comprehensive study of the kinetic properties of the variant enzyme was made. The novel feature of the kinetics is the presence of substrate inhibition by the purine bases, with a true Kᵢ value for hypoxanthine of 80± 20μM, and a normal value for the true Kₘ. The pattern of substrate inhibition is characteristic of that associated with the formation of a dead-end complex and double inhibition experiments indicate that the form of this complex is enzyme-hypoxanthine-PPi. These unusual kinetic properties provided an opportunity to study the order of substrate binding in a way not possible for the normal enzyme and showed an ordered sequential reaction mechanism. Some limited protein-structural studies were performed and showed an altered electrophoretic mobility for the variant enzyme in non-denaturing gels. Secondly, the purine metabolic pathways in cultured cells, derived from T.K., from a patient with the Lesch-Nyhan syndrome, and from normal individuals, were studied. The cells were labelled with precursors of the de novo or of the salvage pathways, usually in the presence of a reference label, and sometimes in the presence of inhibitors of the various steps in the purine metabolic pathways. Hypoxanthine salvage was about 10% of that of control cultures. The growth of cells in a variety of selective media was also studied. Thirdly, as physician in charge of T.K., I could monitor the progress of his hyperuricaemia and observe the effects of therapy throughout the duration of this project.
Open Access
Molecular phylogenetics and conservation aspects of antelopes
(1996) Rebholz, Wilhelmus Ewald Reinaard; Harley, Eric H
This thesis concerns the molecular phylogenetics of three tribes of the family Bovidae, the Antilopini, Neotragini, and Tragelaphini. None of these tribes have been studied extensively with molecular techniques. The tribe Antilopini is one of the most speciose tribes (it includes 6 genera with 20 species) and the classification of several species of the genus Gazella is not clear. The tribe Neotragini is thought to be paraphyletic. Mitochondrial sequences of the cytochrome c oxidase ill and cytochrome b genes totalling 1083 base pairs have been determined for 52 taxa and used to determine phylogenetic relationships using cladistic and distance methods. Karyological analysis identified polymorphisms in several species (especially in Gazella saudiya and G. subgutturosa). Karyotypes of G. dorcas pelzelni and an XXY karyotype of a G. dorcas individual are shown for the first time. The main conclusions are that the Antilopini and the Tragelaphini are monophyletic and that the tribe Neotragini is paraphyletic. There is a lack of phylogenetic resolution between tribes which is probably due to the rapid radiation of the different tribes about 20 million years ago. The genus Taurotragus in the tribe Tragelaphini is shown to be paraphyletic and it would be appropriate to incorporate these taxa in the genus Tragelaphus. The genus Gazella could be paraphyletic, due to the position of Antilope cervicapra, in which case the genus needs to be split into two genera or renamed as Antilope. It is also argued that the use of the subgenus Trachelocele should be discontinued and that its only species, G. subgutturosa should be included in the subgenus Gazella. G. rufifrons and G. thomsonii may be more appropriately considered as conspecific. Cytogenetic and sequence data reveal that the herd of G. saudiya in Al Areen Wildlife Park is hybridised with G. bennettii and it is argued that it is important to identify unhybridised G. saudiya in other collections, since this species is on the brink of extinction. This case study demonstrates the need to genetically screen individuals which are part of a captive breeding program, especially if they are intended for reintroduction into the wild.
Open Access
A molecular phylogeny of the subfamily Arundinoideae (Poaceae)
(1995) Barker, Nigel Paul; Linder, H Peter; Harley, Eric H
The subfamily Arundinoideae has long been considered to be an unnatural assemblage of genera, the relationships of which are obscure or unknown. Because morphological and anatomical data have, to date, been unable to elucidate relationships among these genera, sequence data from two chloroplast genes are used to elucidate relationships among 33 arundinoid genera. Sequence data from the variable, grass-specific insert in the rpoC2 gene is used to determine the relationships among 73 grass species from all currently recognised subfamilies. Phylogenetic analysis of this sequence data required the development of specialised alignment techniques based on testing assumptions of positional homology. Results of the analyses based on these alignments suggest that the Arundinoideae is divisible into four lineages, corresponding approximately to the tribes Danthonieae, Arundineae, Aristideae and Thysanolaeneae. Several arundinoid representatives are placed in other subfamilies. The rpoC2 sequence data was too variable to elucidate relationships at the tribal and subfamilial level. For this purpose, sequence data of the highly conserved rbcL gene was obtained from 22 taxa selected from the lineages identified by the rpoC2 study. Phylogenetic analysis of a total of 36 sequences resolved some of the relationships of the major clades, but other relationships were poorly supported. In an attempt to improve the resolution of these major clades, the rpoC2 and rbcL data sets were combined with restriction site data. These three data sets were analysed in a variety of combinations using both data combination and tree consensus methods to assess support of the phylogenetic relationships. Despite this, the resolution of the relationships among the Arundineae, Danthonieae, Aristideae and Chloridoideae was not resolved with any finality, although a (Arundineae (Danthonieae (Aristideae, Chloridoideae))) relationship is proposed as being most likely. The molecular phylogeny implies that eight grass subfamilies should be recognised. Two of these, the Danthonioideae and Aristidoideae, are new and the Arundinoideae is redelimited. Furthermore, new tribes in the subfamilies Centothecoideae (Thysanolaeneae) and Chloridoideae (Centropodieae) are proposed to accommodate lineages and taxa misplaced in the subfamily Arudinoideae as previously delimited.
Open Access
The molecular systematics of Southern African Testudinidae
(1998) Varhol, Richard Joseph; Harley, Eric H
Sixteen of the world's 42 species of land tortoises occur in Africa, 10 of which are endemic to southern Africa. South Africa itself, which occupies 0.8% of the earth's total land mass, has the highest tortoise biodiversity in the world, with 13 species. This is the first study to use molecular techniques to investigate the evolutionary history of this group, which displays an unusually high level of speciation on the continent. Four hundred and fifty base pairs of mtDNA cytochrome b sequence were obtained, using direct PCR-based sequencing, from 32 individual tortoise blood samples, comprising 13 different species from 6 genera. PAUP 3. 1.1, and MEGA were used to infer a phylogeny using Chrysemys scripta elegans (an Emydid) an outgroup. Both phenetic and cladistic methods generated similar results. With the exception of Malacochersus, both morphological and molecular work show largely congruent results. When intra-specific relationships, using the molecular results, were compared to the existing morphological data, Psammobates was the only genus with a consistent topology. Proposals for the re-evaluation of Homopus, Kinixys and Geochelone have been made. Suggestions, based on molecular results, include the distinction between Chersobius and Homopus (Hewitt 1937), incorporating Malacochersus tornieri into Kinixys, and the elevation of Geochelone pardalis pardalis and G.p. babcocki to species level. Sequencing a further nine individuals within Homopus areolatus showed a higher than expected sequence variation, suggesting a distinct population structure and possibly cryptic species.
Open Access
Systematic relationships in southern African Francolins as determined from mitochondrial DNA
(1991) Jakutowicz, Mariola Barbara; Harley, Eric H
The Francolins constitute the largest genus in the Galliform family Phasianidae. There is little accord concerning the taxonomic classification of its members. In the past, information on this group has been provided by morphological and palaeontological evidence. An investigation into the molecular history of this group is presented, using mitochondrial DNA (mtDNA) as an evolutionary tool. A comparison of mtDNA restriction fragment lengths has been used to help define the phylogenetic relationships between 13 southern African Francolin species and a selected outgroup, the Japanese Quail. Both cladistic and distance-based analytical methods have been used to construct phylogenies from the molecular fragment data. The trees relating the Francolins are in general agreement with the traditional classification based on morphological, behavioural and morphometric studies, but differ in the branching order of two species, F. levaillantii and F. hartlaubi. A recent proposal for the partitioning of the genus into two monophyletic assemblages of quail-like "partridges" and pheasant-like "francolins" is supported by mtDNA fragment data, with the exception of the two aberrant taxa. On the basis of the initial fragment size comparison, F. hartlaubi and F. levaillantii constitute part of an unresolved quadrichotomy at the base of the tree. A restriction endonuclease site mapping approach has been utilized to provide a deeper resolution for the molecular phylogeny. Detailed mtDNA restriction endonuclease maps of F. levaillantii, F. hartlaubi, two species representing the "partridge" and "francolin" monophyletic groups respectively, and also of the Madagascar Partridge, have been constructed. Phylogenetic analysis of this data has helped to resolve the problematic placement of the two aberrant taxa by showing an early separation of F. levaillantii from the "partridge" lineage, and of F. hartlaubi from the "francolin" lineage. The Madagascar Partridge was anticipated to be a likely sister-taxon to the whole group, but instead appears to have close relationships within the "partridge" lineage.
Open Access
Systematics of cetaceans using restriction site mapping of mitochondrial DNA
(1992) Ohland, Derek Paul; Harley, Eric H
A phylogenetic study of eleven cetaceans was undertaken using Restriction Endonuclease Maps (RSM) of mitochondrial DNA (mtDNA). One species from the suborder mysticeti (baleen whales) was sampled, and of the ten odontocetes (toothed whales) sampled two were from the family Ziphiidae (beaked whales) and eight were from the family Delphinidae (dolphins) (each representing a different genus). The primarily opportunistically obtained (i.e. from strandings or accidental death in commercial trawl nets) heart tissue generally yielded high quantities of mtDNA which is needed for double digest fragment analysis. The mtDNA extracted from the sampled taxa was cleaved with fifteen different six-base Restriction Enzymes (RE's). Using the three-way method of analysis and aided by the computer program Resolve (Ver. 2.7) (Harley, unpublished), RSM's were constructed. Distance (Neighbor-Joining and Fitsch-Margoliash) and cladistic (Maximum Parsimony and Bootstrap) methods were used to infer phylogenies. The baleen whale was used as an outgroup for the cladistic analysis. Both the distance and both the cladistic methods produced the same single topology, which is concordant with morphologically based classifications. The two differences (within the Delphinidae), viz. Grampus' most basally rooted position and Cephalorhynchus' grouping with the Delphininae are of taxa whose groupings are unresolved in the morphologically based classifications. Using Brown et al's (1979) molecular clock, very recent divergence times at the generic, family and suborder levels were obtained, when compared to fossil based estimates. Using the odontoceti/mysticeti split the base substitution rate of cetacean mtDNA was estimated to be much slower than that of terrestrial mammals (0,3% compared to 1,0% Myr⁻¹). A similarly slow rate was calculated for cetacean nuclear DNA (nDNA) (0,09% Myr⁻¹) (Schlotterer et al, 1991). It remains an unresolved issue as to whether the base substitution rate of cetacean DNA is slower than terrestrial mammals or whether the fossil evidence needs to be reinterpreted. The time of the mysticeti/odontoceti split is palaeontologically uncertain and the suggested monophyletic status of the extant suborders has been questioned, thus making the calculation of cetacean base substitution rate risky. Equally, the incomplete fossil record can lend itself to misinterpretation.
Open Access
Uric acid metabolism in the Dalmatian coach hound
(1982) Briggs, Oliver Martin; Harley, Eric H
The Dalmatian coach hound, when compared to other dog breeds, exhibits three characteristic abnormalities of uric acid metabolism, namely hyperuricaemia, hyperuricosuria and increased renal uric acid clearance. These properties are associated with hypoallantoinaemia and hypoallantoinuria. A result of these abnormalities is a high incidence of urate urolithiasis in this breed. Other diseases such as recurrent dermatitis, chronic cystitis and deafness are also found in the Dalmatian and whether there is any causal relationship with the uric acid disorder is unknown. In terms of the quantity of uric acid excreted, the Dalmatian dog resembles man more closely than the non-Dalmatian. On the other hand, in its high renal urate clearance, this breed of dogs differs from man, whose renal clearance values are lower and therefore closer to those of the non-Dalmatian. In this respect the Dalmatian resembles man affected with the inborn renal urate transport defect of renal hypouricaemia. The significance of uric acid metabolism in the Dalmatian has attracted many investigators in search of the underlying metabolic defect(s). Study of the Dalmatian may also be relevant to the understanding of disorders of purine metabolism in man for example hyperuricosuria, uric acid urolithiasis and hereditary renal hypouricaemia.
Open Access
The use of cultured cells with defects of citrulline metabolism in diagnosis and in the study of intercellular communication
(1985) Davidson, James Schonland; Harley, Eric H
Citrullinemia and argininosuccinic aciduria are two disorders resulting from defects in two consecutive enzymes of the urea cycle, argininosuccinate synthetase and argininosuccinate lyase. Fibroblast cell lines were derived from patients with these disorders and the diagnoses, which had been made on the basis of amino acid levels in plasma and urine, were confirmed by demonstrating that the cell lines were unable to incorporate 14 c-citrulline into protein. DNA from the argininosuccinate synthetase-deficient (ASS⁻) cells was analysed by restriction enzyme digestion and hybridisation to a cDNA probe which had been cloned from human argininosuccinate synthetase mRNA. No defect in the patient's DNA could be demonstrated, indicating that no major deletions in the argininosuccinate synthetase genes were present in this patient. Co-cultures of the ASS⁻ and argininosuccinate lyase-deficient (ASL⁻) fibroblasts were able to incorporate 14 citrulline into protein at rates comparable to normal fibroblasts. This complementation did not require cell fusion, was dependent on cell contact, and was not the result of exchange of metabolites or enzymes via the culture medium. These results indicated that complementation occurred by the exchange of metabolites via intercellular junctions between the two cell types. Co-cultures of ASS⁻ and ASL⁻ cells were used as an assay system for measuring intercellular junctional communication. This allowed quantitation of the effects of pH and extracellular divalent cations on junctional communication. Tumor promoters such as phorbol esters and organochlorine pesticides have been reported to inhibit intercellular junctional communication in other systems, and this inhibitory activity may be related to the mechanism of tumor promotion. The organochlorine pesticide 1,1,1-trichloro- 2,2-bis(p-chlorophenyl)ethane (DDT) was shown to be an inhibitor of junctional communication in ASS⁻/ASL⁻ cocultures. This inhibition was reversible, of rapid onset, and independent of extracellular calcium. The tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13- acetate (TPA) also rapidly induced inhibition of junctional communication. However, co-cultures between Chinese hamster V79 cells, which are deficient in ASS⁻, and ASL⁻ human fibroblasts were more sensitive to inhibition by TPA than the original ASS⁻/ASL⁻ co-cultures. Refractoriness to TPA occurred following prolonged treatment with high concentrations of TPA. Retinoic acid and other retinoids also inhibited junctional communication, and the inhibitory effects of retinoic acid and TPA were additive. The significance of these results in relation to the anti-tumor-promoting activity of retinoic acid is discussed. It is concluded that co-cultures of ASS⁻ and ASL⁻ cells constitute a useful system for providing quantitative measurements of intercellular junctional communication under a wide range of experimental conditions.

Browsing by Author "Harley, Eric H"

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