Biomedical subjects
Publications and source records attributed to P Nygaard.
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The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.
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If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2-14C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-14C]thymidine is not associated with DNA. The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases. Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic. The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.
Specular microscopy helps to estimate the viability of the corneal endothelium long before clinical signs of decompensation occur. Therefore, specular microscopy is useful for the selection of eyes for lens implantation in general; for the prognosis of the eye with a lens implant; and for the evaluation of the method of cataract surgery, of lens implantation techniques, and possibly of lens design, material, and manufacturing. A long-term study of intracapsular and extracapsular pseudophakic eyes revealed that the intracapsular pseudophakic eyes had significantly less endothelial cells, which could partly be explained by toxicity of the Supramid used as loop material, but mostly by assuming continuing endothelial disintegration in the intracapsular eyes. In 41 patients our extracapsular technique caused an average cell loss of only 6.7%.
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The principle and technique of Maurice's method of microscopy of the corneal endothelium at high magnificant is discussed. An instrument is now available for clinical use (clinical specular microscope). The experience of the authors in patients with an intraocular lens is reported. A rather high deficit of endothelial cells was found in a series of 26 children and young adults, that had suffered injury from 2 to 11 years previously. An average immediate endothelial cell loss of only 6-7% was found in extracapsular cataract extraction with simultaneous implantation of a 2-loop or 4-loop intraocular lens. A striking difference was found between the two eyes of bilateral pseudophakic patients when one eye had undergone intracapsular surgery and the other eye extracapsular surgery. It had to be concluded that barrier deprivation of the intracapsular aphakic eye leads to continuing endothelial cell loss other than through ageing. Subclinical and clinical late corneal dystrophy therefore belong to the "barrier deprivation syndrome" together with retino-vascular accidents (macular and optic disc oedema) and vitreous degeneration.
The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli. The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed. It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.
Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.
The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.