PubMed Health⌕ Search

Biomedical subjects

J D Karkas

Publications and source records attributed to J D Karkas.

At least 37 records · Page 2Linked to original sources
PubMed1985

Synthesis and antiherpetic activity of (S)-, (R)-, and (+/-)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine, linear isomers of 2'-nor-2'-deoxyguanosine.

Racemic 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(+/-)-iNDG], a new analogue of acyclovir (ACV) and a structural analogue of 2'-nor-2'-deoxyguanosine (2'NDG), was synthesized and found to inhibit the replication of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequently, its optical isomers, (R)- and (S)-iNDG, were prepared from chiral intermediates. The chloromethyl ethers of 1,2-di-O-benzyl-D- and -L-glycerol were made and reacted with tris(trimethylsilyl)guanine to give the 9-alkylated guanines, which were deprotected by catalytic hydrogenolysis. Against HSV-1 and HSV-2 in cell culture, (S)-iNDG was approximately 10- to 25-fold more active than the R enantiomer and had an ED50 comparable to those for ACV and 2'NDG. The inferior activity of (R)-iNDG paralleled the poor inhibition of viral DNA polymerase by its phosphorylation products. In mice infected intraperitoneally or orofacially with HSV-1 or intravaginally with HSV-2, (S)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(S)-iNDG] was less efficacious than 2'NDG but comparable to or more active than ACV.

Acyclovir↗
PubMed1983

A comparison of the antiviral agents 2'-nor-2'-deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates.

A comparative study was conducted between the antiherpetic agents 2'-nor-2'-deoxyguanosine (2'NDG) and acyclovir (ACV) with respect to 1) the relative rates of uptake and phosphorylation to the "active" triphosphate species in tissue culture and 2) the in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates. The results indicated that a) six hours after HSV1 infection of primary rabbit kidney cells there was seven times more 2'NDG-triphosphate in the cells than ACV triphosphate; b) the relative rate of triphosphate formation in HSV1-infected versus uninfected cells was 4.5 times higher for 2'NDG than for ACV and c) the triphosphate of 2'NDG (2'NDG-TP) was a more selective inhibitor of the viral compared to the cellular DNA alpha-polymerase than the triphosphate of ACV (ACV-TP). The Km/Ki ratios for 2'NDG-TP and ACV-TP (in the competitive inhibition of dGTP) were 3.10 and 1.37, respectively, for the highly purified HSV1 polymerase; and 0.05 and 1.11, respectively, for the partially-purified HeLa alpha-polymerase. Neither triphosphate inhibited the HeLa DNA beta-polymerase to any significant extent. These results are in line with the findings [Ashton et al. (1982), Biochem. Biophys. Res. Commun. 108, 1716-1721] that 2'NDG has superior in vivo antiherpetic activity compared to ACV without apparent toxicity.

Acyclovir↗
PubMed1983

9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication.

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (2'-nor-2'-deoxyguanosine; 2'NDG) selectively inhibits the replication of herpes group viruses. In cell culture studies 2'NDG was at least 10-fold more potent than acyclovir (ACV) in inhibition of human cytomegalovirus replication and Epstein-Barr virus-induced lymphocyte transformation and was about as effective as ACV in inhibition of herpes simplex viruses 1 and 2 and varicella zoster virus. Orally administered 2'NDG was 6- to 50-fold more efficacious than ACV in treating systemic or local HSV-1 infection or HSV-2 intravaginal infection in mice. The mode of action of 2'NDG appears to involve phosphorylation by herpes simplex virus thymidine kinase and subsequent phosphorylations by cellular kinases to produce 2'NDG triphosphate, which is a potent inhibitor of herpes virus DNA polymerase. Compared to ACV, 2'NDG was a more efficient substrate for HSV-1 thymidine kinase (Vmax/Km for 2'NDG 30-fold higher than that of ACV), whereas 2'NDG monophosphate is a more efficient substrate for GMP kinase (Vmax/Km for 2'NDG monophosphate 492-fold higher than that for ACV monophosphate). The combined effect is more rapid production of the inhibitory triphosphate from 2'NDG than from ACV.

Acyclovir↗
PubMed1975

A DNA polymerase from embryos of Drosophila melanogaster. Purification and properties.

The more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.

Ammonium Sulfate↗
PubMed1973

Reverse transcription by Escherichia coli DNA polymerase I.

E. coli DNA polymerase I (EC 2.7.7.7) can engage in either DNA- or RNA-directed DNA synthesis with hybrid templates. The choice of the strand to be transcribed depends primarily on the relative lengths of the two strands of the hybrid, the longer strand serving as the template and the shorter as the primer. If a polynucleotide is reduced in size by exposure to an endonuclease before being hybridized to the complementary strand, the template efficiency of the latter increases several-fold. Under properly selected conditions, highly efficient reverse transcription of the all-ribonucleotide template-primers poly(A).oligo(U), poly(C).oligo(I), and poly(I).oligo(C) can be achieved. "f1 RNA," the RNA strand of an f1 DNA.RNA hybrid, can also serve as template for reverse transcription either after "nicking" of the hybrid with DNase, or after separation from the DNA strand and priming by DNase-treated f1 DNA.

Adenine Nucleotides↗
PubMed1972

Action of DNA polymerase I of Escherichia coli with DNA-RNA hybrids as templates.

Experiments indicating the ability of the ribo strand of a DNA-RNA template to guide polydeoxynucleotide synthesis by highly purified DNA polymerase I of E. coli (EC 2.7.7.7) are presented. With poly(rA).poly(dT) as template, poly(dT) is formed with a high efficiency, but almost no poly(dA). The specific activity of the enzyme, when tested with this template under suitable conditions, is eight times greater than that found for the poly(dA-dT) template. Single-stranded DNA fractions, with no template activity for DNA polymerase, are converted to efficient templates after their transcription by RNA polymerase. A concerted polymerization reaction, in which the action of DNA polymerase is dependent on that of RNA polymerase, can also be demonstrated with synthetic polydeoxynucleotides and single-stranded fractions of denatured DNA as templates.

Adenine Nucleotides↗
PubMed1972

DNA polymerase of chicken embryo: purification and properties.

The almost complete purification of the previously reported DNA polymerase of chicken embryo is described; in the final isolation step the enrichment of the enzyme, which comprises about 0.01% of the protein extractable from 10-day-old embryos, is more than 9000-fold. The enzyme is a basic protein (pI = 9.15), of molecular weight about 27,000. It exhibits no nuclease activity, requires Mn(2+) ion for activity, and may contain Zn. It is very sensitive to oxidation.

Ammonium Sulfate↗
PubMed1972

Coupling of replication to transcription in vitro.

In a coupled system consisting of RNA polymerase and DNA polymerase I of Escherichia coli, the four deoxyribo- and the four ribonucleoside triphosphates, and DNA of bacteriophage f1 as template, DNA synthesis depends on the concomitant synthesis of RNA. Over a wide range of concentrations of the two polymerases, RNA synthesis was unaffected by the simultaneous synthesis of DNA, whereas the rate of DNA synthesis depended on the level of RNA synthesis. In the coupled reaction, RNA synthesis starts immediately at a high rate, which subsequently decreases, whereas DNA synthesis starts after a lag and its rate increases as the reaction proceeds. Upon addition of rifampicin, the rate of RNA synthesis falls abruptly, while that of DNA declines only gradually. The base composition of the DNA synthesized in the coupled reaction is complementary to that of f1 DNA template. It is suggested that the RNA synthesized by the RNA polymerase serves as a primer rather than as a template for the DNA polymerase.

Base Sequence↗
PubMed1972

Mechanism of DNA replication by highly purified DNA polymerase of chicken embryo.

Highly purified DNA polymerase of chicken embryo was used to investigate the mechanism of DNA replication. The templates used included synthetic homopolymer pairs, as well as DNA of bacteriophage f1 and DNA-RNA hybrids prepared by partial or complete transcription of f1 DNA. The evidence suggests that the DNA synthesized is complementary either to the DNA or the RNA strand of the hybrid, depending on the relative lengths of the two strands, with the longer serving as the template and the shorter as the primer. While f1 DNA itself lacks template properties, the f1 DNA-RNA hybrids are efficient templates; and composition studies show the DNA synthesized to be complementary to the DNA strand of the hybrid. These observations suggest a mechanism of DNA replication in which the initial synthesis of a stable primer by RNA polymerase plays a pivotal role.

Adenine Nucleotides↗
PubMed1971

Nucleic acid polymerases of the developing chicken embryo: a DNA polymerase preferring a hybrid template.

This paper presents a preliminary survey of the nucleic acid polymerases of the developing chicken embryo, especially of the 4-day stage. The predominant activity is that of a DNA polymerase preferring a DNA-RNA hybrid as the template. The enzyme, which is activated by Mn(2+) ions and inhibited by p-chloromercuriphenylsulfonate, copies preferentially the ribo strand of a hybrid, such as poly(rA).poly(dT), but is relatively inactive with all-ribo duplexes. DNA polymerase I of Escherichia coli was also found to use the hybrid template with high efficiency, copying preferentially the ribo strand. With the chicken enzyme, the template activity of denatured DNA was increased tenfold by simultaneous transcription with RNA polymerase. DNA polymerase activity reaches a maximum in the 6- to 8-day chicken embryo and then declines progressively to about one-third of the maximal value in the adult chicken.

Adenine Nucleotides↗
PubMed1970

Template properties of complementary fractions of denatured microbial deoxyribonucleic acids.

DNA preparations from seven bacterial species and from E. coli phage T4, and also the complementary L and H fractions into which these DNA specimens, after denaturation, were separated by chromatography on methylated albumin-kieselguhr columns, were studied as templates in the RNA polymerase system, and the nucleotide composition of the RNA products was determined. The RNA transcripts of the separated L and H fractions were found to be faithful copies of the respective DNA fractions. This suggests "transcription analysis" as a sensitive and reliable analytical technique for the determination of the base composition of denatured DNA. The L and H fractions of T4 DNA were shown, both by temperature-absorbance profiles and by transcription analysis, to be mutually complementary. The RNA products formed with intact DNA as the template were not exact copies of the latter; their composition indicated that under our experimental conditions the "heavy" DNA strand is transcribed preferentially.

Adenine↗
PubMed1969

Separation of microbial deoxyribonucleic acids into complementary strands.

DNA preparations from seven bacterial species and from the E. coli phage T4 can, after denaturation with alkali, be separated chromatographically into two distinct components (L and H) through intermittent gradient elution from methylated albumin kieselguhr columns. The direct chemical analysis of the L and H fractions isolated from DNA specimens of the AT type shows them to exhibit a high degree of complementarity; but despite a bias in the distribution of purines and pyrimidines, either fraction contains equimolar quantities of 6-amino and of 6-keto nucleotides. In the L and H components derived from DNA of the equimolar and GC types, the distribution bias appears limited to guanine and cytosine. It is suggested that the L and H fractions represent the complementary DNA strands.

Bacillus↗