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G R Reddy

Publications and source records attributed to G R Reddy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular characterization of a 28 kDa surface antigen gene family of the tribe Ehrlichiae.

Antisera against different Ehrlichiae recognize an immunodominant, cross-reacting approximately 28 kDa surface antigen defined as the MAP1 in Cowdria ruminantium. These antigens are considered valuable in developing serodiagnostic tests and recombinant vaccines for Ehrlichiae infections. To evaluate the relationship in three closely related Ehrlichiae, Ehrlichia chaffeensis, Ehrlichia canis, and C. ruminantium, the structure of the 28 kDa antigen genes was analyzed. We describe the cloning and characterization of DNA encoding genes homologous to MAP1 from E. chaffeensis and E. canis. The cloned segment of E. chaffeensis contains one expressed and four transcriptionally silent tandemly arranged, nonidentical genes; the E. canis locus consists of two nonidentical genes. Comparative analysis of these genes revealed the presence of four conserved regions separated by three highly variable regions. B-cell epitope analysis identified three major cross-reacting epitopes that map to the variable regions. Location of the epitopes at the variable regions and the presence of multigene family with only one expressed copy suggest a mechanism of immune evasion in these Ehrlichiae.

Amino Acid Sequence↗
PubMed1998

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber.

We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs. We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column. Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate. The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX. When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences.

Arachidonate 5-Lipoxygenase↗
PubMed1997

Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde.

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-alpha]purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. M1G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(M1G)TCCG-3' into a gapped-duplex derivative of the vector. Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G-->A and M1G-->T mutations with few M1G-->C mutations. The total mutation frequency was approximately 1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication. The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

DNA Adducts↗
PubMed1997

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay.

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of > 99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.

Animals↗
PubMed1997

Analysis of the malondialdehyde-2'-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/negative chemical ionization/mass spectrometry.

A method is described for the assay of the major malondialdehyde-deoxyguanosine adduct (M1G) based on immunoaffinity purification and gas chromatography/electron capture/negative chemical ionization/mass spectrometry. A stable isotope of M1G-deoxyribose ([2H2]M1G-dR) was used as an internal standard. Recovery of internal standard throughout the entire assay procedure was approximately 40%. The assay showed a linear response over a range of 10-1000 pg of M1G-dR and was verified by analysis of a synthetic. M1G-containing oligomer. The limit of detection in biological samples was 100 fmol/sample, corresponding to 3 adducts/10(8) bases for 1 mg of DNA. DNA was isolated from the blood of 10 healthy human donors, and M1G levels were measured. A mean value of 6.2 +/- 1.2 adducts/10(8) bases was obtained, with no obvious differences bases on age or cigarette smoking. A small, but statistically significant difference was observed between the levels in females (5.1 +/- 0.4 adducts/10(8) bases) and males 6.7 +/- 1.1 adducts/10(8) bases). The presence of M1G in leukocyte DNA was further verified by analysis using liquid chromatography/electrospray ionization mass spectrometry.

Adult↗
PubMed1997

Temperature-dependent formation of a conjugate between tris(hydroxymethyl)aminomethane buffer and the malondialdehyde-DNA adduct pyrimidopurinone.

The stability of the major adduct formed between the endogenous product malondialdehyde (MDA) and deoxyguanosine, a pyrimidopurinone termed M1G-dR, was tested under a variety of conditions required for nucleic acid manipulation. M1G-dR was found to be stable at neutral pH and 37 degrees C but to be unstable when stored at -20 degrees C in the presence of Tris buffers. A new product with a characteristic absorption band at 350 nm was identified by 1H-NMR as an enamino-imine comprised of one molecule of Tris, one molecule of MDA, and deoxyguanosine. The formation of the conjugate was observed on reaction of Tris with M1G-dR or its ring-opened derivative N2-(3-oxo-1-propenyl)deoxyguanosine. The Tris-M1G-dR conjugate was unstable in aqueous solutions at room temperature, undergoing hydrolysis. However, the Tris conjugate of M1G base remained stable at room temperature in organic solvent. The isolation and properties of a conjugate between M1G-dR and Tris suggest that cross-links may form by reaction of MDA with DNA but they are likely to be unstable to hydrolysis.

Cross-Linking Reagents↗
PubMed1996

Inhibition of proliferation and of IL-2 production and utilization in lymphocytes by S-oxalylglutathione.

Previously we have shown that S-oxalins (monothiolesters of oxalic acid) are ubiquitous mammalian metabolites whose concentrations decrease when lymphocytes are stimulated to proliferate. The present study was undertaken to further examine the role of S-oxalins in the proliferation process. When added to lymphocytes stimulated with concanavalin A, the S-oxalin, S-oxalylglutathione (GS-Ox), inhibited DNA synthesis by 50% when present at ca. 0.15 mM and virtually 100% at 0.5 mM. The inhibition was reversible. The presence of GS-Ox blocked IL-2 production, but addition of IL-2 did not permit DNA synthesis to proceed. GS-Ox also inhibited proliferation of an IL-2-dependent cell line, BT2. In primary lymphocytes GS-Ox reduced IL-2 receptor expression, but not in an IL-2-dependent blast cell line. Overall RNA synthesis and protein synthesis were not significantly altered by GS-Ox. Levels of the positive transcription factor, NF-kappaB, were decreased after incubation of lymphocytes with GS-Ox, but the amount of a negative transcription factor, NREA, was largely unchanged. The results not only provide further evidence that S-oxalins are small-molecule cell proliferation inhibitors, they also clarify to some extent the specific steps in the activation response modulated by S-oxalins.

Animals↗
PubMed1996

Current status of the Plasmodium falciparum genome project.

The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.

Animals↗
PubMed1996

Characterization of an N6-oxopropenyl-2'-deoxyadenosine adduct in malondialdehyde-modified DNA using liquid chromatography/electrospray ionization tandem mass spectrometry.

Malondialdehyde (MDA), a product of lipid peroxidation, causes mutations in bacterial and mammalian cells and cancer in rats. MDA reacts with deoxynucleosides in vitro and the monomeric adduct of MDA with deoxyguanosine (M1G-dR) is the major adduct. M1G-dR has been detected in rat and human liver. Random mutagenesis studies with MDA-modified DNA and recent 32P-postlabeling studies indicate that in addition to M1G-dR, adducts to deoxyadenosine may also be formed. We have utilized liquid chromatography coupled with electrospray ionization tandem mass spectrometry to characterize an N6-oxopropenyl-2'-deoxyadenosine adduct (M1A-dR) in calf DNA modified with MDA.

Animals↗
PubMed1996

Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas.

The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan.

Africa↗
PubMed1996

Monitoring Babesia bovis infections in cattle by using PCR-based tests.

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.

Animals↗
PubMed1995

Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frame shift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics.

Structural refinement from solution 1H NMR data was performed on the 5'-d[ATCGC(PdG)-CGGCATG]-3'.5'-d[CATGCCGCGAT]-3' duplex, in which the adducted oligodeoxynucleotide containing the exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was annealed with the complementary strand which contained a CpG deletion. The resulting duplex required PdG and one adjacent cytosine to be unpaired. A total of 352 distances were utilized to restrain molecular dynamics calculations, of which 264 were NOE-derived. These distances were calculated using complete relaxation matrix methods from hybrid matrices, which were comprised of the experimentally determined distances and additional distances derived from either A-form or B-form DNA. A simulated annealing protocol combined with the distance restraints was able to refine a single structure with an average rms deviation of < 1.35 A. The accuracy of the refined structure was assessed using full relaxation matrix calculations, which gave good agreement with measured NOE intensities. PdG was found to be stacked into the helix below base pair C3.G18, whereas C5 was found to be unpaired and extruded toward the major groove and parallel to base pair G6.C17. This created a localized bend in the DNA helix of approximately 20-35 degrees at the junction between PdG and C5. The bending corroborated previous assays performed on this modified sequence [Moe, J. G., Reddy, G. R., Marnett, L. J., & Stone, M. P. (1994) Chem. Res. Toxicol. 7, 319-328].

Base Sequence↗
PubMed1995

A repetitive DNA probe for the sensitive detection of Fasciola hepatica infected snails.

Epizootiologic studies on F. hepatica frequently use microscopic techniques for the detection of infected snails, however, the poor efficiency, sensitivity, and specificity associated with these techniques limit their usefulness. A DNA-based test for the identification of snails infected with larval stages of F. hepatica would solve these problems and enable a level of detection accuracy previously unavailable. We have cloned and sequenced a 124 bp fragment of repetitive DNA from F. hepatica which hybridizes specifically with DNA of F. hepatica but not with DNA of its snail intermediate hosts Fossaria cubensis and Pseudosuccinea columella, or with DNA of Fascioloides magna and Paramphistomum liorchis, ruminant trematodes which share the same intermediate host and same enzootic range as F. hepatica. Using this 124 bp fragment as a probe, infection in snails was detected immediately following miracidial penetration, thus a sensitivity equivalent to the minimum biologic unit of the parasite was achieved. This 124 bp repeated sequence belongs to a large family of 124 bp repeats that share a high level of sequence identity and constitute approximately 15% of the F. hepatica genome. We also report here the development of a quick and inexpensive DNA extraction protocol for use in field-collected snails. Thus, we have developed both a highly sensitive and specific DNA probe and a means to use the probe in a large epizootiologic study of F. hepatica where thousands of field-collected snails need to be assayed for infection.

Animals↗
PubMed1995

Cloning and characterization of a Plasmodium falciparum cyclophilin gene that is stage-specifically expressed.

An immunosuppressive agent, cyclosporin A (CsA), has antimalarial activity in several Plasmodium species. Cyclophilins of several species including Plasmodium falciparum exhibit peptidyl-prolyl cis-trans isomerase activity which is inhibited by CsA. A gene encoding P. falciparum cyclophilin (PFCyP) was cloned and characterized. This gene has the entire coding sequence for the mature protein plus a 39-amino-acid-long N-terminal extension. Most of the amino acids predicted to be involved in the peptidyl-prolyl cis-trans isomerase activity and CsA binding are present in the cloned gene. The PFCyP also has the single highly conserved tryptophan residue that is a major determinant in the inhibition of PPIase activity by CsA. The PFCyP coding sequence with or without the N-terminal amino-acid extension was used to construct recombinant expression vectors which were transformed into E. coli. Both vectors produced enzymatically active mature PFCyP proteins that were sensitive to CsA. Northern blot analysis of RNA isolated from the synchronized parasite cultures verified the expression of PFCyP in all erythrocytic stages of the parasite, but at variable levels. The highest level of expression was observed in ring-stage parasites, a stage shown to be more susceptible to CsA. Inhibition of P. falciparum growth in vitro by CsA was re-evaluated for chloroquine-sensitive and chloroquine-resistant strains of the parasite. Essentially, there was no difference between the two strains for the concentration of CsA required to yield 50% inhibition in 48 h of exposure (0.25-0.4 microM).

Amino Acid Isomerases↗
PubMed1995

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

Amino Acid Sequence↗
PubMed1995

Characterization of arachidonate 12-lipoxygenase found in the liver of mongrel dog and its immunohistochemical localization in neutrophils.

The cytosol fraction of non-parenchymal cells isolated from the liver of adult mongrel dogs converted arachidonic acid to 12S-hydroxy-5,8,10,14-eicosatetraenoic acid. The arachidonate 12-lipoxygenase enzyme reacted with linoleic and alpha- and gamma-linolenic acids as well as arachidonic acid. The enzyme was immunoprecipitable with an antibody against the leukocyte 12-lipoxygenase, but not with an antibody against the platelet 12-lipoxygenase. By immunohistochemical observation with anti-leukocyte 12-lipoxygenase antiserum, hepatocytes, Kupffer cells and sinusoidal endothelial cells were not immunostained, but a large number of neutrophils located in sinusoidal cavities were positively stained. The 12-lipoxygenase activity thus detected may be attributed predominantly to the neutrophils appearing in sinusoidal cavities rather than the non-parenchymal cells in the liver of mongrel dog upon infection. In agreement with this finding there was essentially no neutrophil accumulation in the liver of Beagle dog which had no bacterial and parasitic infection.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver.

Endogenous DNA adducts may contribute to the etiology of human genetic disease and cancer. One potential source of endogenous DNA adducts is lipid peroxidation, which generates mutagenic carbonyl compounds such as malondialdehyde. A sensitive mass spectrometric method permitted detection and quantitation of the major malondialdehyde-DNA adduct, a pyrimidopurinone derived from deoxyguanosine. DNA from disease-free human liver was found to contain 5400 adducts per cell, a frequency comparable to that of adducts formed by exogenous carcinogens.

Adolescent↗