Biomedical subjects

M Fry

Publications and source records attributed to M Fry.

At least 19 recordsLinked to original sources

PubMed1992

Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80).

The site of action of the antimalarial compound 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80), would appear to be the mitochondrial respiratory chain. Studies reported herein have demonstrated 566C80 to be a potent and selective mitochondrial inhibitor with mitochondria isolated from Plasmodium falciparum and P. yoelii. Selective assay of individual respiratory chain complexes has shown the primary site of action of 566C80 to be the cytochrome bc1 complex (Complex III): supportive evidence from difference spectroscopy indicates the site of inhibition to lie between cytochromes b and c1 of this complex. Using [14C]566C80, evidence is presented which suggests that 566C80 may become irreversibly bound to a polypeptide with an approximate molecular mass of 11,500 Da.

Animals↗

PubMed1992

A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion.

In this study we examined whether the arrest of DNA polymerase alpha (pol alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis.

Base Sequence↗

PubMed1992

Subcellular fractionation of the two organelle DNAs of malaria parasites.

Malaria parasites contain two extrachromosomal DNAs, a 6 kb repetitive linear molecule which is assigned on the basis of its genetic content to the mitochondria, and a 35 kb transcriptionally active circular molecule whose intracellular location is not known. We used the polymerase chain reaction to detect and estimate the numbers of both molecules in sub-cellular fractions derived from the rodent parasite Plasmodium yoelii. The two DNA molecules were not coordinately partitioned by the fractionation process, the 6 kb molecule being more abundant, relative to the 35 kb circle, in a fraction enriched for mitochondria, the converse being true for a less dense fraction of unknown identity. This implies that the two molecules are located in different cellular compartments, and is consistent with other evidence suggesting they have different evolutionary origins.

Animals↗

PubMed1991

Mitochondria of mammalian Plasmodium spp.

Highly purified mitochondrial fractions have been isolated from the intraerythrocytic stages of two mammalian Plasmodium spp., Plasmodium yoelii of rodents and Plasmodium falciparum of man. Mitochondria of the former parasite are cristate whereas those of the latter are essentially acristate. Isolated mitochondria from both parasite species were heterogeneous with respect to size, shape, density of matrix staining and extent of internal structure. Respiratory assay, by reduction of exogenous cytochrome c, showed NADH, alpha-glycerophosphate and succinate to be the substrates with the greatest potential for metabolism. Additionally, proline, dihydroorotate and glutamate (P. falciparum only) were oxidized at low rates. A number of NAD(+)-linked substrates were not utilized. The NADH-dependent reduction of cytochrome c was insensitive to rotenone and antimycin A. Fumarate inhibited the NADH-dependent reduction of cytochrome c and stimulated the oxidation of NADH, suggestive of an NADH-fumarate reductase pathway. Oxidation of either alpha-glycerophosphate or succinate was fully inhibited by standard mitochondrial electron transport inhibitors, including a number of Complex III inhibitors, although the concentrations required of such inhibitors (notably myxothiazol) were relatively high compared to mammalian mitochondria. Dithionite-reduced minus oxidized difference spectra indicated the presence of cytochromes aa3, b, c and c1 in mitochondria of both parasite species, but at a higher cytochrome to protein ratio in P. yoelii. Freshly isolated mitochondria from either species exhibited only low respiratory control ratios with alpha-glycerophosphate or succinate as substrates. The apparent absence of a respiratory chain 'Site I' in such mitochondria may mean that NADH-fumarate reductase serves to reoxidize mitochondrial NADH.

Animals↗

PubMed1991

Atrial repair for transposition of the great arteries: current approach in Zürich based on 24 years of follow-up.

Long-term survival after atrial repair of a transposition of the great arteries between 1964 and 1988 is analyzed in 254 thirty-day survivors. For the complete series, overall survival is 99.6% after one year, 94.9% after 6 years, 89.6% after 12 years, 84.9% after 18 years, and 64.0% after 24 years. Broken down for simple (154/254) versus complex (95/254) transposition survival, it is 99.4% versus 100.0% for one year, 94.7% versus 99.5% for 6 years, 90.8% versus 88.3% for 12 years, 85.1% versus 85.8% for 18 years and 77.7% versus 46.8% at 24 years. Survival for patients without or with VSD is 95.3% versus 96.2% after 6 years, 91.2% versus 87.9% after 12 years, 87.5% versus 84.0% after 18 years, and 83.7% versus 52.5% after 24 years. Cumulative survival as a function of the surgical approach for VSD closure is 94.3% for transatrial versus 92.3% for transventricular at 6 years, 82.2% versus 92.3% after 12 years, 82.2% versus 83.0% after 18 years and 82.2% versus 41.5% after 24 years. Survival for patients operated upon before 1978 is 94.3% versus 96.0% thereafter at 6 years, and 90.7% versus 89.2% at 12 years (NS). Linearized mortality rates are 1.49% per patient year for the complete series, 0.93 for simple transpositions, 2.21% for complex transpositions (including VSD, obstruction of the left ventricular outflow tract, or both), 0.68% for transpositions without VSD, 1.98% for transpositions with VSD, 2.43% for transposition with transventricular repair of a VSD, 1.74% for transposition with transatrial closure of a VSD, and 0.90% for all cases operated upon after 1978.(ABSTRACT TRUNCATED AT 250 WORDS)

Actuarial Analysis↗

PubMed1991

Endogenous mutations and cancer.

We have analyzed two aspects concerning the relationship between mutations and cancer. Firstly, we consider the possibility that spontaneous mutations result from normal cellular processes. Errors in DNA replication, depurination of DNA, and damage to DNA by oxygen free radicals are important potential sources of endogenous mutations. Mutations produced by DNA polymerases in vitro are clustered, and consist predominantly of single-base substitutions. Analysis of termination sites during copying by DNA polymerase-alpha indicates that certain pause sites are associated with misincorporation of single non-complementary nucleotides, and thus pause sites could be associated with mutagenic hotspots. Depurination results predominantly in transversion mutations resultant from the incorporation of deoxyadenosine opposite abasic sites. Mutations resulting from oxygen free radical damage to DNA are predominantly single-base substitutions: C- > T, G- > C, and G- > T being the most frequent. In addition, there is an unusual mutation that results from oxygen free radical damage to DNA, two adjacent cytidines being substituted by two thymidines. The frequency of mutations observed in tumors appears to be much greater than that predicted from the known rates for spontaneous mutagenesis. Recent observations suggest that many human tumors contain four or more independent mutations. If these multiple mutations are each required for tumor development, then the occurrence of malignancies based on normal mutation rates would be exceedingly rare, and it may be necessary to postulate that tumors result from an increase in the rate of endogenous mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts.

The genome of human immunodeficiency virus (HIV) and especially the envelope gene are mutated with unusually high frequency during in vivo replication. Recent studies indicate that HIV reverse transcriptase (RT) is unusually error prone and that the number of generated mutations is disproportionately high within repetitive base sequences. To study the ability of recombinant and wild-type HIV RT to traverse specific homo-oligomeric stretches, we used bacteriophage M13 DNA templates that contain different oligo(purine) and oligo(pyrimidine) inserted tracts. The progress of HIV RT along these templates was potently inhibited from further progression only at a (dA)16 insert. Comparison with other polymerases indicates that the almost complete blockage of polymerization beyond an oligo(dA) insert is unique to HIV RT and Moloney murine leukemia virus RT, which has high sequence homology with HIV RT. The extent of termination of HIV RT at the oligo(dA) run is not affected by alterations in the concentration of KCl, Mg2+, dNTP, or by a decrease in pH. Obstruction of HIV RT opposite the oligo(dA) insert is not alleviated by moving the primer position further upstream from the oligo(dA) insert. Lastly, HIV RT purified directly from virions is also specifically arrested at an oligo(dA) tract. Competition experiments indicate that the concentration of active HIV RT in the presence of M13(dA)16 DNA is similar to that observed in the presence of M13(dG)16 DNA. In addition, preincubation of M13(dA)16 DNA with HIV RT does not subsequently inhibit avian myeloblastosis virus RT from successfully traversing the (dA)16 insert. Therefore, it appears that the blockage of chain elongation of HIV RT at the (dA)16 insert is not the result of trapping the enzyme at this site.

Avian Myeloblastosis Virus↗

PubMed1990

Protracted alanine aminotransferase levels in tamarins infected with hepatitis A virus.

Eighteen red-bellied tamarins (Saguinus labiatus), experimentally infected with hepatitis A virus (HAV), were followed for up to 1.5 years after initial challenge. Half of these animals developed protracted alanine aminotransferase (ALT) abnormalities, which lasted for between 23 and 55 weeks post-challenge. IgM anti-HAV was detected intermittently during the early phase of their relapsing hepatitis and never after return of ALT levels to normal. The possibility that these findings may be related to continued HAV replication, coinfection with another agent, or the result of an autoimmune phenomenon triggered by HAV are discussed.

Alanine Transaminase↗

PubMed1990

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and oligomers of prostaglandin B1.

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and their oxidative metabolites and/or polymers was studied using partially purified human phospholipases A2 and [1-14C]oleate labelled, autoclaved E. coli as substrate. As previously reported for other phospholipases A2, oleic and arachidonic acids inhibited human synovial fluid phospholipase A2 with IC50s of 15 and 30 microM respectively. Air oxidation of arachidonic acid or hydroxylation of oleic acid (12-hydroxy-oleate) substantially relieved that inhibition. Similarly, the enzymatically oxidatized metabolite of arachidonate, prostaglandin B1 (PGB1), did not inhibit enzymatic activity. However, prostaglandin Bx (PGBx), an oligomer (n = 6) of PGB1, was a potent inhibitor of Ca(++)-dependent, neutral-active phospholipase A2 activities. Enzymatic activity in acid extracts from human neutrophils, platelets, sperm, plasma, synovial fluid, endometrium, degenerative disc, and snake venom was inhibited by PGBx with IC50s ranging from 0.5-7.0 microM. Inhibition was independent of substrate phospholipid concentration over a 24-fold range (5-120 microM) and PGBx quenched the tryptophan fluorescence of snake venom phospholipase A2 in a dose-dependent manner. Agonist-induced (A23187) release of arachidonic acid from prelabelled human neutrophils and cultured human endothelial cells was also inhibited by PGBx with IC50s of 3 and 20 microM, respectively. These results illustrate that oxidative reactions of cis-unsaturated fatty acids relieve their natural inhibitory activity, and polymerization of an inactive fatty acid metabolite yields a potent inhibitor of in vitro and in situ phospholipase A2 activity.

Arachidonic Acid↗

PubMed1990

Nippostrongylus brasiliensis and Ascaridia galli: characterization of peroxisomes.

A method is described for the isolation of peroxisomes from mitochondria-enriched fractions obtained from both species of nematodes. The distributions of these organelles are characterized after density gradient centrifugation in sucrose or Percoll by urate oxidase and catalase activities. The possession of peroxisomes may be part of an important defence mechanism in parasites.

Animals↗

PubMed1990

Effect of mitochondrial inhibitors on adenosinetriphosphate levels in Plasmodium falciparum.

1. The effects of mitochondrial inhibitors on the ATP levels of intraerythrocytic Plasmodium falciparum have been studied. 2. Changes in parasite ATP or ADP levels with time in response to various mitochondrial inhibitors appear quite complex; ATP levels may be initially depressed and then elevated above normal, but the nature of the response depends upon the stage in the intraerythrocytic cycle and in some cases upon the concentration of the inhibitor used. 3. After ca 2 hr incubation of cultures with inhibitors ATP levels appear to be stabilized and are similar to those of untreated parasites. However, ADP levels of trophozoites show significant increases after a 2 hr incubation with inhibitors, particularly with oligomycin and to a lesser extent with antimycin A; increases in ADP levels however were not observed in ring-stages of the parasite. 4. Inhibition of red cell and parasite glycolysis leads to rapid decreases in parasite ATP levels which are not significantly affected by oligomycin. Incubation of in vitro cultures with oligomycin can result in a decreased, rather than increased rate of lactate production with a concomitant appearance of pyruvate in the growth medium. 5. This investigation would indicate that if there is a mitochondrial contribution to the parasite ATP pool it is relatively small, and that a short-fall in this contribution is quickly compensated for by ATP from other source(s), although this is not necessarily met by increased glycolysis.

Adenosine Diphosphate↗

PubMed1989

Sequence specificity of pausing by DNA polymerases.

We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines). Each of these inserts hinders the advancement of the large fragment of E. coli Pol I during DNA synthesis. The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH. Blockage is not affected by either the concentration of template or by the position of the DNA primer. The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point. There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures. Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase. This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide.

Base Sequence↗

PubMed1989

Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases.

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.

Adenosine Monophosphate↗

PubMed1989

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases.

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.

Animals↗

PubMed1989

Diferric transferrin reductase in Plasmodium falciparum-infected erythrocytes.

Diferric transferrin reductase, assayed by formation of ferrous - bathophenanthroline, is detectable in the plasmamembrane of intact mature erythrocytes infected with Plasmodium falciparum. The absence of this transmembrane redox system in uninfected mature erythrocytes suggests its synthesis and incorporation by the intraerythrocytic parasite. The presence of diferric transferrin reductase, together with parasite-derived transferrin-receptor in the erythrocyte membrane, suggests a transferrin receptor-mediated uptake of iron by the malaria parasite.

Animals↗

PubMed1989

Studies of GB hepatitis agent in tamarins.

Three tamarins (Saguinus labiatus), two of which had previously been infected with hepatitis A virus and parenteral non-A, non-B hepatitis, were inoculated intravenously with the agent of GB hepatitis. All three animals developed alanine aminotransferase abnormalities 2 weeks after inoculation. Peak alanine aminotransferase levels were recorded 4 weeks postinoculation. These declined thereafter but continued to fluctuate at abnormal levels for 32 weeks. Liver biopsies showed liver cell swelling and inflammation with focal necrosis. Portal tracts and areas around central veins were heavily infiltrated with mononuclear cells. A fourth animal (no previous exposure to hepatitis viruses) inoculated with GB was killed on Day 15 postinoculation. Serum and extracts of liver and feces from this day were used as inocula for three other animals. Only the serum and liver extract transmitted GB hepatitis. The fecal specimen did not transmit and a fecal extract taken at a later date from another animal was also noninfectious. GB hepatitis virus is distinct from the viruses causing Type A and blood-borne non-A, non-B-hepatitis. Although the virus is present in serum and has previously been transmitted per os, it is not shed in feces.

Alanine Transaminase↗

PubMed1988

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.

Animals↗

PubMed1988

Haemoprotein terminal oxidases in the nematodes Nippostrongylus brasiliensis and Ascaridia galli.

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.

Animals↗