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N J Leonard

Publications and source records attributed to N J Leonard.

At least 19 recordsLinked to original sources
PubMed1995

Analysis of chromosomal abnormalities in human sperm after chemotherapy by karyotyping and fluorescence in situ hybridization (FISH).

The frequency of numerical and structural chromosomal abnormalities was studied in the sperm of a lymphoma patient 3 years after MACOP-B chemotherapy (CT). Sperm karyotyping was performed by fusion of human sperm with hamster oocytes and analysis of 193 Q-banded sperm chromosomes. Multicolor fluorescence in situ hybridization (FISH) was performed on 10,228 sperm for analysis of disomy frequencies for chromosomes 1 and 12 and on 10,664 sperm for chromosomes X and Y. Sperm karyotyping demonstrated numerical abnormalities in 7.3% of the spreads, 6.7% hypohaploid and 0.5% hyperhaploid, giving a conservative estimate of aneuploidy of 1%. Structural chromosomal abnormalities were present in 7.3% of the karyotypes and 0.5% had both numerical and structural abnormalities. Results of FISH analyses yielded disomy frequencies of 0.10%, 0.11%, 0.04%, 0.05%, and 0.18% for chromosomes 1, 12, X, Y, and XY, respectively. The frequency of diploid sperm was 0.09%. The frequency of abnormalities was not significantly increased compared to control donors for any of the studies. Also, the frequencies of X- and Y-bearing sperm did not differ significantly from 50% in the sperm karyotyping or FISH studies.

Aneuploidy↗
PubMed1994

Preparation and characterization of polyclonal and monoclonal antibodies specific for covalently linked DNA/RNA cross sections.

Covalently linked cross sections refer to structures that mimic hydrogen-bonded purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine duplexes. Cross sections dA [symbol:see text] U and A [symbol: see text] dT, which have been synthesized chemically, have molecular dimensions similar to purine-pyrimidine base pairs in a double helix. We propose that antibodies to such covalent cross sections might facilitate the study of the pathogenesis of specific diseases or of biochemical processes in which base pair involvement is suspected and/or demonstrated. We have made polyclonal antibodies against "A:U" and "A:T" cross sections by immunizing rabbits with dA [symbol: see text] U and A [symbol: see text] dT, each conjugated to keyhole limpet hemocyanin (KLH). The antibodies were found to be highly specific for the cross sections and to cross react minimally to single nucleosides. Hybridomas secreting monoclonal antibodies to "A:T" were then generated from spleen cells of mice immunized with A [symbol: see text] dT conjugated to KLH. The MAbs produced were also found to be highly specific for "A:T" among various nucleosides. In fact, the binding of most of the monoclonal antibodies to "A:T" was only partially inhibited by high concentrations of adenosine or thymidine. All monoclonal antibodies to "A:T" cross react, but with lower affinity, to "A:U." Selected MAbs showed greater inhibition of binding to "A:T"-BSA by A + T than by A or T alone.

Adenine↗
PubMed1994

The raspberry locus of Drosophila melanogaster includes an inosine monophosphate dehydrogenase like coding sequence.

DNA from the raspberry gene of Drosophila melanogaster has been cloned through P-element tagging of a dysgenically induced lethal allele. A transcribed DNA segment adjacent to the P insert encodes an amino acid sequence that is similar to known inosine monophosphate dehydrogenase (IMPDH) sequences. Further dysgenically induced lethals and all four known spontaneous ras eye-colour mutations have changes in the DNA either within or just upstream from the transcribed region. Identification of IMPDH as a probable product of the ras gene is compatible with the finding of an allele that requires dietary guanosine (GR), since the enzyme mediates the first of two biosynthetic steps leading from inosine monophosphate (IMP) to guanosine monophosphate (GMP). However, other auxotrophic mutants at the locus remain unexplained by the finding. The results further suggest that GMP synthesis from IMP is an essential process, despite the capacity of the fly for salvage synthesis of GMP from GR. Consideration of the phenotypes associated with mutants at the ras locus suggests that IMPDH activity is regulated in a tissue-specific manner.

Alleles↗
PubMed1992

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway.

Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.

Adenosine Triphosphatases↗
PubMed1991

Ligation with T4 RNA ligase of an oligodeoxyribonucleotide to covalently-linked cross-sectional base-pair analogues of short, normal, and long dimensions.

Compounds that are covalent analogues of nucleic acid base pairs of normal, long, and short C1' to C1' dimensions [B. Devadas and N.J. Leonard (1990) J. Am. Chem. Soc., 112, 3125-3135.] have been added to the oligodeoxyribonucleotide d(A)6 with bacteriophage T4 RNA ligase as a prelude to placing them at defined loci within nucleic acid duplexes. Analogue cross sections that represent a normal Watson-Crick base pair as well as a pyrimidine-pyrimidine and a purine-purine apposition were ligated in modest yields (approximately 20%) to the oligonucleotide. Ligation conditions were optimized for each analogue, and the cross section was joined to only a single oligonucleotide in each case. The structures of the ligated products were proved by HPLC, enzymatic degradation, and spectroscopic analyses.

Chromatography, High Pressure Liquid↗
PubMed1991

Which 3-ribofuranosyl-substituted purine 5'-phosphates undergo template-directed oligomerization?

We have studied the oligomerization reactions of the 2-methylimidazolide derivatives of 3-isoisoguanosine 5'-phosphate (2) and 3-isoxanthosine 5'-phosphate (5) in the presence of a variety of homopolynucleotide templates. In no case did we observe a substantial template-facilitated production of long oligomers. Polyuridylic acid directed the synthesis of low molecular-weight products from both monomers. Polycytidylic acid, polyadenylic acid, polyinosinic acid, and polyguanylic acid were ineffective as templates in the systems that we investigated.

Chromatography, High Pressure Liquid↗
PubMed1989

Structure of 3-isoinosine.

3-beta-D-Ribofuranosylhypoxanthine, 3-beta-D-ribofuranosyl-1,6-dihydro-3H-purin-6-one, C10H12-N4O5, Mr = 268.23, monoclinic, P2(1), a = 6.503 (1), b = 24.007 (6), c = 7.392 (2) A, beta = 106.53 (2) degrees, V = 1106.3 (9) A3, Z = 4, Dx = 1.610 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 1.23 cm-1, F(000) = 560, T = 299 K, R = 0.048 and wR = 0.043 for 1602 observed reflections. There are two crystallographically independent molecules in the structure; in both of them the 6-oxo, 7H tautomer is the predominant form. The bond lengths and angles of the two molecules are almost identical and the hypoxanthine moiety is almost planar. The torsional angles of the glycosidic linkage O(4')-C(1')-N(3)-C(4) are -159.3 and -148.8 degrees, both in the anti range. The sugar puckers are 4T3 (C4'-exo/C3'-endo), with P = 46.35 degrees and tau m = 42.30 degrees, and 2E (C2'-endo), with P = 157.24 degrees and tau m = 41.32 degrees. All N and O atoms except N(3) and O(4') participate in a three-dimensional hydrogen-bonding system.

Chemical Phenomena↗
PubMed1988

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates.

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1988

Structure of 3-isoadenosine.

3-beta-D-Ribofuranosyladenine, C10H13N5O4, Mr = 267.25, orthorhombic, P2(1)2(1)2(1), a = 13.470 (4), b = 16.054 (6), c = 5.141 (2) A, V = 1111.8 (7) A3, Z = 4, Dx = 1.596 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 1.18 cm-1, F(000) = 560, T = 298 K, R = 0.043, wR = 0.042 for 1136 reflections. The tautomeric form present in the crystal is 6-NH2. The adenine moiety is nearly planar and the torsional angle of the glycosidic linkage O(4')-C(1')-N(3)-C(4) is -161.5 degrees. All N and O atoms except N(3) and O(4') participate in a three-dimensional hydrogen-bonding system.

Adenosine↗
PubMed1988

Template-directed oligomerization of 3-isoadenosine 5'-phosphate.

Template-directed oligomerization of an activated derivative of 3-isoadenosine 5'-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3'-5'-linked oligomers while the reaction of ImpA gives only 2'-5'-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.

Adenosine Monophosphate↗
PubMed1988

Nonenzymatic synthesis of 5-aminoimidazole ribonucleoside and recognition of its facile rearrangement.

5-Amino-1-beta-D-ribofuranosylimidazole 5'-monophosphate (AIR, 1) is the ubiquitous precursor to the purine ribonucleotides in vivo, and it serves as the biochemical precursor to the pyrimidine portion of thiamin (vitamin B1) in certain prokaryotic organisms. The corresponding ribonucleoside (AIRs, 5b) was prepared via chemical (nonenzymatic) synthesis from 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide. The tri-O-acetylated derivative of AIRs (5a) was also prepared, and it was shown to undergo a facile ring transformation in aqueous pH 7 buffer to afford N-(imidazol-4-yl)-2,3,5-tri-O-acetyl-D-ribofuranosylamine as a 1:2 mixture of alpha and beta anomers (6a). Under similar conditions, compound 5b affords the corresponding unprotected beta-ribonucleosides 6b. This Dimroth-type ring transformation reaction of 5 to 6, which occurs primarily in neutral aqueous solution, may be responsible for the previously reported lability of AIRs and its derivatives. It may also have relevance to the postulated early biotic pathway to the 9- and 3-substituted purine nucleotide components of an all-purine biopolymer.

Acetylation↗
PubMed1985

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides.

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.

Animals↗
PubMed1984

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: nucleotide binding to the chemically modified catalytic subunit.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates the catalytic subunit of the adenosine cyclic 3',5'-monophosphate dependent protein kinase isolated from bovine cardiac muscle by covalent modification of lysine-71, whereas 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with cysteines-199 and -343 to inactivate the enzyme. All three of these reagents have been postulated to modify residues at or near the active site of the catalytic subunit. ATP (2 mM) in the presence of excess Mg2+ (10 mM) protects the enzyme against inactivation by these reagents. AMP did not afford any protection, but adenosine slightly decreased the rate of inactivation. The specific effects of covalent modification of lysine-71 and cysteines-199 and -343 on nucleotide binding were characterized by fluorescence-polarization titrations with lin-benzoadenine nucleotides as fluorescent ligands. lin-Benzoadenosine is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (38 microM) similar to the Ki for adenosine (35 microM). This value agrees well with the Kd (32 microM) for adenosine determined by fluorescence-polarization titrations. lin-Benzoadenosine 5'-diphosphate (lin-benzo-ADP) has been shown to be a competitive inhibitor with respect to ATP [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], and lin-benzoadenosine 5'-triphosphate (lin-benzo-ATP) is a substrate for the phosphotransferase activity of the protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗