PubMed Health⌕ Search

Biomedical subjects

H Rodriguez

Publications and source records attributed to H Rodriguez.

At least 55 records · Page 3Linked to original sources
PubMed1997

Genital Chlamydia trachomatis infection in pregnant adolescents in east Tennessee: a 7-year case-control study.

STUDY OBJECTIVE: To examine the prevalence, symptomatology, risk factors, and other infections associated with urogenital chlamydial infection in pregnant teenagers. DESIGN: Retrospective case-control study by medical record review. SETTING: Prenatal care clinic for adolescents at University of Tennessee Medical Center, Knoxville, Tennessee. PARTICIPANTS: Pregnant adolescents younger than 19 years of age who were diagnosed with chlamydial infection on the first prenatal visit from 1988 to 1994 were studied. Pregnant adolescents of similar age and socioeconomic background who came in the same day for the first prenatal visit, but were not infected, made up the control group. INTERVENTION: Routine prenatal questionnaires regarding personal and medical histories, and routine prenatal screening, including pelvic examination with Papanicolaou (PAP) smear and laboratory investigations for common genital infections and sexual transmitted disease (STDs), were obtained. MAIN OUTCOME MEASURES: Analyzed the prevalence of chlamydial infection and compared the infected group to the control group with regard to race, behavioral factors, symptoms, prenatal screening results, other concurrent genital infections, and histories of STDs. RESULTS: Of a total population of 596 pregnant teenagers, 67 (11.24%) were infected with Chlamydia trachomatis. In multivariate analysis, black race (odds ratio [OR] = 4.01; 95% confidence interval [CI] = 1.74-9.23; p = 0.001) and greater gestational age at first prenatal visit (OR = 1.11; 95% CI = 1.04-1.18; p = 0.001) were independently associated with chlamydial infection. Age, marital status, number of pregnancies, smoking, alcohol abuse, drug abuse, age at first intercourse, and multiple sex partners were not associated with the infection. Likewise, the symptom of vaginal discharge (a complaint of > 70% in each group), other genital co-infections (found > 50% in each group, mainly candidiasis and bacterial vaginosis), abnormal PAP smears (found > 60% in each group) and histories of STDs or previous chlamydial infection were not significantly different between case and control groups. Human papillomavirus infection, trichomonal infection, and dysplasia or atypia were found more often in patients infected with chlamydia, but were not statistically significant. CONCLUSION: Pregnant adolescents in east Tennessee were at risk for chlamydial infection as well as for other genital infections and abnormal PAP smears. Routine prenatal chlamydial screening is warranted because of a lack of specific symptoms.

Adolescent↗
PubMed1997

Transcription of the human genes for cytochrome P450scc and P450c17 is regulated differently in human adrenal NCI-H295 cells than in mouse adrenal Y1 cells.

Human NCI-H295 cells, which express all of the genes for the steroidogenic enzymes in a hormonally regulated fashion, should be an ideal system in which to study the transcriptional regulation of these genes. Using deletional promoter/reporter constructions for the human P450scc and P450c17 genes, we identified the regions conferring basal and cAMP-induced transcription of these two genes in NCI-H295 human adrenal cells. In the P450scc gene, both basal and cAMP-induced transcriptional activation elements lie within the first 79 bp upstream (-79) from the transcriptional start site. In the P450c17 promoter, both basal and cAMP-responsive elements lie within the first upstream 63 bp, and a second basal element lies between -184 and -206 bp. The locations of these elements are substantially different from the locations of elements that appear to be functionally equivalent when these human gene promoters are transfected into mouse adrenal Y1, mouse testicular MA-10, or human choriocarcinoma JEG-3 cells. These data indicate that the transcriptional regulation of these genes in their native species and cell type differs substantially from their regulation in cells from other species and tissues, and suggests that the results from transfection experiments examining genes for steroidogenic enzymes in heterologous cells may not reflect events in vivo.

Adrenal Glands↗
PubMed1996

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications.

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Ascorbic Acid↗
PubMed1996

Gene conversion in the CYP11B2 gene encoding P450c11AS is associated with, but does not cause, the syndrome of corticosterone methyloxidase II deficiency.

Cytochrome P450c11AS (aldosterone synthase) has 11 beta-hydroxylase, 18-hydroxylase, and 18-oxidase activities and is expressed solely in the adrenal zona glomerulosa. Corticosterone methyloxidase II (CMOII) deficiency denotes a rare disorder of adrenal steroidogenesis in which only the 18-oxidase activity of P450c11AS is disrupted, while the 11 beta-hydroxylase and 18-hydroxylase activities persist. Such patients have elevated serum concentrations of corticosterone and 18-hydroxycorticosterone and very low or unmeasurable concentrations of aldosterone, often resulting in a clinical salt-losing crisis in infancy. One pair of point mutations, Arg181-->Trp and Val386-->Ala, has been previously characterized to cause this disorder in an inbred Iranian Jewish population. We have sought mutations causing CMOII deficiency in outbred populations. In three of four unrelated P450c11AS alleles from two unrelated patients with CMOII deficiency, we found a gene conversion event in which exons 3 and 4 of the CYP11B2 gene encoding P450c11AS were changed to the sequence of the nearby CYP11B1 gene, which encodes the related enzyme P450c11 beta. This conversion resulted in a mutant P450c11AS protein carrying three changes: Asp141-->Glu, Lys151-->Asn, and Ile246-->Thr. We built seven vectors expressing P450c11AS carrying each mutation singly, each of the three possible pairs of mutations, and the triple mutation as found in the proband. The activities of both the normal P450c11AS and the various mutants in transfected nonsteroidogenic COS-1 cells were very low, but their activities in steroidogenic MA-10 and JEG-3 cells were 10- to 20-fold higher. In these systems all of the mutants retained normal 18-oxidase activity, indicating that the detected gene conversion event is associated with but does not cause CMOII deficiency. None of the four CYP11B2 alleles in these two patients bore other identifiable mutations. These patients might have mutations in the promoters or other noncoding regions, or mutations in genes other than CYP11B2 may cause the syndrome of CMOII deficiency.

Base Sequence↗
PubMed1996

Genetic variation in P450c11AS in Chilean patients with low renin hypertension.

Low renin hypertension (LRH), which accounts for 10-20% of patients with idiopathic "essential" hypertension, bears hormonal similarities to mineralocorticoid-induced hypertension, but elevated mineralocorticoid concentrations have not been found. Some patients with LRH have normal, rather than suppressed, plasma aldosterone concentrations, so that the ratio of aldosterone concentration to PRA (Aldo/PRA) is high, suggesting inappropriately increased aldosterone biosynthesis. We characterized the CYP11B2 gene that encodes the aldosterone synthase, P450c11AS, in hypertensive and control populations in a single clinic in Santiago, Chile. We directly sequenced the entire CYP11B2 gene in 12 patients with LRH, 2 high renin hypertensive controls, and 2 normotensive controls. All sequences were identical, except that 8 of 24 LRH alleles encoded arginine rather than lysine at position 173. The Arg173 and Lys173 variants were expressed in transfected MA-10 cells, and their ability to convert deoxycorticosterone to aldosterone was measured; the apparent Michaelis constant (Km) for Lys173 was 2.73 mumol/L; the Km for Arg173 was 2.53 mumol/L. The apparent maximal velocity (Vmax) for Lys173 was 6.5 x 10(-3) micrograms/mL.24 h; the Vmax for Arg173 was 7.8 x 10(-3) micrograms/mL.24 h. The first order rate constant, Vmax/Km was 2.38 for Lys173 and 3.08 for Arg173. As these values were not significantly different, we sought to determine whether Arg173 is a polymorphism linked to LRH. We examined position 173 in 52 unselected patients with idiopathic hypertension and 55 normotensive controls by PCR amplification of CYP11B2 exons 3-5 followed by digestion with Bsu361, which digests the Arg173 sequence, but not the Lys173 sequence. More of the hypertensive alleles (39 of 104, 37.5%) than normotensive alleles (25 of 110, 22.5%) carried Arg173 (chi 2 = 5.57; P < 0.02). Most of the Arg173 alleles (31 of 72, 43.1%) were from hypertensive patients with Aldo/PRA below 30, whereas only 5 of 24 (20.8%) Arg173 alleles were found in patients with Aldo/PRA greater than 30 (chi 2 = 3.79; P = 0.05) Thus, the ARg173 variant of CYP11B2 may be linked to LRH in Chilean patients.

Adult↗
PubMed1996

Seasonality and freezability vs routine parameters in stallion semen.

The fertilizing ability of stallion semen was analyzed using fresh and frozen samples, obtained before (June-July) or during (October-November) the breeding season. Thirty ejaculates obtained from 4 stallions were used. The analysis comprises routine seminogram; ATP concentration (Comhaire et al., 1983); subjective and objective motility and sperm velocity (Makler, 1980). Freezing was done following the technique of Martin et al. (1979). Sperm velocity, ATP content and objective motility in ejaculates of subjective motility >50% show values of 14.0 + or - 0.84 mu m s(-1); 4.8 + or - 2.7x10(-7)M and 54.0 + or - 7.4%, respectively. For ejaculates with subjective motility <50%, these values are 8.4 + or - 2.4; 0.74 + or - 0.36x10(-7)M and 27.0 + or - 0.8%. No significant changes in these characteristics were elicited by freezing, though ATP content dropped to 50% after thawing. These characteristics are highly associated between them (p<0.05) and with some conventional parameters of the routine seminogram such as sperm motility, count, total spermatozoal number and morphology. Additionally, sperm chromatin packing was analyzed by decondensing sperm nuclei using a thiol reducing agent. This parameter was not modified by freezing and it may depend on prolonged epididymal sperm storage during the non-breeding season. Epididymal maturation then results in an excessive disulphur bridging in sperm basic proteins, so that hypermaturation with faulty male pronuclear formation can follow.

Adenosine Triphosphate↗
PubMed1995

Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.

Adrenal Cortex↗
PubMed1995

Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction.

The ligation-mediated polymerase chain reaction was used to map the frequency of reactive oxygen species-induced DNA damage at nucleotide resolution in genomic DNA purified from cultured human male fibroblasts. Damaged pyrimidine and purine bases were recognized and cleaved by the Nth and Fpg proteins from Escherichia coli, respectively. Strand breaks and modified bases were induced in vitro by copper ion-mediated reduction of hydrogen peroxide in the presence of ascorbate; reactant concentrations were adjusted to induce lesions at a frequency of 1 per 2-3 kilobases in purified genomic DNA. Glyoxal gel analysis demonstrated that the ratio of induced strand breaks to induced base damage was 0.8/2.7 in DNA dialyzed extensively to remove adventitious transition metal ions. Ligation-mediated polymerase chain reaction analysis of the damage frequency in the promoter region of the transcriptionally active phosphoglycerate kinase (PGK 1) gene revealed that (Cu(II)/ascorbate/H2O2 caused DNA base damage by a sequence-dependent mechanism, with the 5' bases of d(pGn) and d(pCn) being damage hot spots, as were the most internal guanines of d(pGGGCCC) and d(pCCCGGG). Since base damage occurs after formation of a DNA-Cu(I)-H2O2 complex, these data suggest that the local DNA sequence affects formation of DNA-Cu(I)-H2O2 complexes and/or the efficiency of base oxidation during resolution of this complex.

Base Sequence↗
PubMed1995

Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene.

(+)-anti-B[a]PDE-induced mutagenesis is being investigated, including in a supF gene of the E. coli plasmid pUB3. Based upon various findings a working hypothesis was proposed that the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is able to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT vs. GC-->CG) depending upon its conformation in DNA, which can be influenced by various factors, such as DNA sequence context. Frameshift mutations are also significant and are analyzed herein. In virtually all cases one of three possibilities is observed: (1) some treatments change frameshift and base substitution mutation frequency (MF) in a quantitatively parallel fashion; (2) other treatments, which change frameshift MF, can change base substitution MF in a quantitatively reciprocal fashion; finally, (3) there are treatments that do not change frameshift MF, and also do not change base substitution MF. (Changes can be brought about by SOS induction, differing DNA sequence context, or heating adducted pUB3 prior to transformation. Why different kinds of changes result in (1) vs. (2) vs. (3) is discussed.) Thus, base substitution and frameshift mutagenesis pathways appear to be coupled in some way, which is most easily rationalized if both pathways are interrelated. The simplest mechanism to rationalize this coupling is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base substitution pathway. The surprising implication is that--although different conformations are likely to be required to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT; see above)--a single conformation can give rise to either a base substitution or a frameshift mutation. Frameshift and base substitution pathways must eventually diverge, and it is proposed that this is controlled by factors such as DNA sequence context.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Aldosterone↗
PubMed1995

Transcriptional regulation of P450scc gene expression in neural and steroidogenic cells: implications for regulation of neurosteroidogenesis.

Regulation of steroidogenesis in classic endocrine tissues is mediated by transcriptional regulation of the P450scc gene, which encodes the first and rate-limiting cholesterol side-chain cleavage enzyme. We previously showed that P450scc messenger RNA is regionally expressed in the adult rat brain, primary glial cultures, and C6 glioma cells. Expression of P450scc in the brain results in the de novo synthesis of neurosteroids, a class of steroid hormones that are active at gamma-aminobutyric acidA and N-methyl-D-aspartate receptors. We determined whether P450scc expression is transcriptionally regulated in neural cells, using the same DNA sequences and nuclear proteins as classic steroidogenic adrenal and Leydig cells. The transcriptional activity of deletional mutants of 2.5 kilobases of the 5'-flanking regulatory region of the rat P450scc gene cloned into a luciferase reporter gene was assessed in mouse adrenocortical Y-1, mouse Leydig MA-10, rat C6 glioma, rat GC somatotrope, and mouse GT1-7 neurosecretory cell lines. P450scc was transcriptionally regulated in Y-1, MA-10, and C6 glioma cells, but not in GC or GT1-7 cells. In one region (-94/-35), putative steroidogenic factor-1-binding sites appeared to be critical for the basal transcriptional activity and cAMP responsiveness in steroidogenic Y-1 and MA-10 cells, but had no function in rat C6 cells. DNA sequences between -94/-130 mediated both basal and cAMP-inducible transcriptional activity in C6 cells. Gel mobility shift assays showed that one nuclear protein binding to DNA sequences between -54 and -35 was abundant in MA-10 and Y-1 cells, but was absent from C6 cells, whereas another nuclear protein, binding to DNA sequences between -94 and -130 was abundant in C6 cells, but was rare in MA-10 cells and absent from Y-1 and other adrenocortical cells. Although the DNA sequence between -94 and -130 contains an Sp1 site, Sp1 did not bind to this site. Nevertheless, this GC-rich region was critical for nuclear protein binding and for basal and cAMP-induced transcriptional regulation in both C6 and MA-10 cells. These observations demonstrate that the rat P450scc gene is transcriptionally regulated in glioma cells, but its regulation in glial cells involves a DNA element different from those used in classic steroidogenic tissues. The results further suggest that steroidogenic factor-1 is not involved in regulating neurosteroidogenesis.

Adrenal Cortex↗
PubMed1995

Mutation T318M in the CYP11B2 gene encoding P450c11AS (aldosterone synthase) causes corticosterone methyl oxidase II deficiency.

Corticosterone methyl oxidase (CMO) deficiency refers to disorders of aldosterone synthesis due to mutations in the CYP11B2 gene encoding cytochrome P450c11AS, which is the adrenal aldosterone synthase. Type I CMO deficiency is associated with low concentrations of 18OH-corticosterone and aldosterone, due to severe mutations in P450c11AS; while type II CMO deficiency is associated with high concentrations of 18OH-corticosterone and low concentrations of aldosterone, due to less severe mutations of P450c11AS. A single type of mutation, compound homozygosity for R181W and V386A, has been reported as the cause of CMOII deficiency in an inbred population. We now report a patient with a typical clinical and hormonal picture of CMOII deficiency. Direct sequencing of patient and parent DNAs showed that the mother's allele contributed R181W and the deletion/frameshift mutation delta C372, while the father's allele contributed T318M and V386A. These mutants were recreated in cDNA expression vectors singly and in the parental pairs, showing that neither allele contributed any measurable activity. This would suggest the patient should have CMOI deficiency. These studies suggest that other factors besides P450c11AS are involved in the genesis of the distinctive CMOI and CMOII phenotypes.

Base Sequence↗
PubMed1994

Follicular dynamics, embryo production, and hormonal responses in Brahman heifers following sympathetic stimulation.

The purpose of this experiment was to characterize adrenal and ovarian responses to N-methyl-beta-phenethylamine (NMP) or ACTH. Thirty-three heifers with functional midcycle corpora lutea were placed on a 4-d superovulation regimen. They were injected twice daily with FSH-P and either saline (Control), ACTH (80 IU), or NMP (1 mg/kg BW). An initial blood collection preceded jugular delivery of saline, NMP, or ACTH, i.m. delivery of FSH and PGF2 alpha (d 3), and ultrasound (d 1 to 4). A second blood collection was made 6 min after treatment. Sampling continued daily until d 13. Embryos (age 6 to 7 d) were collected and evaluated. Concentrations of cortisol (posttreatment minus pretreatment) were greatest (P < .05) in NMP- and ACTH-treated heifers. Treatment did not affect mean numbers of small (< 4 mm), large (> 8 mm), or total follicles on d 2 to 4. Heifers receiving NMP had fewer medium follicles (4 to 8 mm) on d 2 and 3, and ACTH-treated heifers had fewer medium follicles on d 4 (P < .07). Mean estradiol concentrations on d 2 to 4 were unaffected by treatment (P > .32). Following PGF2 alpha, time to onset of standing estrus was less (P < .05) in Control than in ACTH- and NMP-treated heifers (41.4 vs 47.9 and 58.9 h, respectively). A greater frequency (P < .05) of reproductive anomalies (no estrus; no ovulation [i.e., progesterone < 1 ng/mL]; luteal failure [i.e., progesterone < 1 ng/mL but embryos recovered]) occurred in NMP (5/11) than in Control (0/10) or ACTH (2/10) heifers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Mutagenesis by the (+)-anti-diol epoxide of benzo[a]pyrene: what controls mutagenic specificity?

Mutagenesis by (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-B[a]PDE], an important mutagenic/carcinogenic metabolite of benzo[a]pyrene (B[a]P), is being studied in order to understand the factors that influence mutagenesis both quantitatively and qualitatively. A new mutational system, which permits the selection of supF- mutations in an Escherichia coli plasmid, pUB3, was used. The work described herein is an extension of previous work, which involved plasmid adduction and then immediate transformation (Rodriguez & Loechler, 1993), and began with the observation that mutation frequency (MF) decreased approximately 2-fold when the (+)-anti-B[a]PDE-adducted plasmid pUB3 is either (1) frozen and then thawed prior to transformation or (2) heated at 80 degrees C for 10 min prior to transformation. Several results suggest that this decrease is not due to the loss of labile adducts. To begin to understand this phenomenon, the mutagenic spectra are compared for (+)-anti-B[a]PDE in supF for the unheated (187 mutants), the freeze/thawed (134 mutants), and the heated (254 mutants) samples. In general, freeze/thawing and heating cause a decrease in all classes of mutations. Considering substitution mutations at G.C base pairs, which predominate, the mutagenic specificity for the combined data sets is GC-->TA (57%), GC-->AT (23%), and GC-->CG (20%). This raises the question, how does (+)-anti-B[a]PDE generate this complex mutagenic specificity, which contrasts with the situation for, e.g., simple methylating agents? One factor is that mutagenic specificity at a particular guanine residue can be influenced by the base on its immediate 5'-side, most notably where mutations are virtually exclusively restricted to GC-->TA in 5'-TG-3' sequence contexts. One unexpected finding may provide additional insight. G115 in supF, which is the major hot spot for base-pairing mutagenesis, is the only site where the qualitative pattern of mutagenesis is significantly affected by heating the (+)-anti-B[a]PDE-adducted plasmid prior to transformation. Without heating, G115-->T mutations predominate, but following heating there is a statistically significant increase in the fraction of G115-->A and G115-->C mutations. The most likely model to explain this and other results is (1) a particular DNA adduct can adopt multiple conformations, (2) the conformation adopted by an adduct can be influenced by various factors, including DNA sequence context, as well as heating and freeze/thawing, and (3) each of these conformations can cause a different pattern of mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Mutagenic specificity of the (+)anti-diol epoxide of dibenz[a,j]anthracene in the supF gene of an Escherichia coli plasmid.

This study was designed to examine the mutagenic specificity of (+)anti-dibenz[a,j]anthracene 3,4-diol-1,2-epoxide ((+)anti-DB[a,j]A-DE) in SOS-induced repair-proficient Escherichia coli ES87 (delta pro-lac, strA)/F' (pro+, lac1Q, lac1am26, lacZ delta M15). The plasmid pUB3, which contains the mutation target gene, supF, was modified with (+)anti-DB[a,j]A-DE in vitro (two to five adducts/plasmid) and then transformed into bacteria by electroporation. The spontaneous mutation frequency for unmodified pUB3 in uninduced cells was about 2 x 10(-6) and for SOS-induced cells, about 8 x 10(-6). The spontaneous supF- mutations were primarily insertions, deletions, and frameshifts. The mutation frequency for (+)anti-DB[a,j]A-DE-modified pUB3 was about 8 x 10(-6) and about 32 x 10(-6) for uninduced cells and SOS-induced cells, respectively. (+)anti-DB[a,j]A-DE induced primarily point mutations in supF in SOS-induced cells. GC-->AT transitions were the major mutations observed in SOS-induced cells (37%). GC-->TA (21%) and GC-->CG (8.6%) transversion mutations were also observed, whereas mutations at AT base pairs were rare (1.9%). Furthermore, a large number of tandem GC/GC-->AT/AT transition mutations were also observed (about 15% of all mutations in SOS-induced cells). Taken together, single and tandem GC-->AT mutations accounted for slightly over half (about 51%) of the mutations observed in SOS-induced cells. These results demonstrated that (+)anti-DB[a,j]A-DE was mutagenic in repair-proficient E. coli; however, unlike other polycyclic aromatic hydrocarbons that induce primarily transversion mutations, (+)anti-DB[a,j]A-DE caused mostly GC-->AT transitions.

Base Sequence↗
PubMed1993

Using hormone-treated pregnant cows as a potential source of oocytes for in vitro fertilization.

In vivo collection of oocytes during pregnancy may be alternative method of obtaining gametes for in vitro fertilization (IVF) from genetically superior gestating cattle. The objectives of this experiment were to induce follicular growth in mature beef cows during each trimester of pregnancy, and then to collect oocytes and verify oocyte competency by IVF and subsequent embryo culture in vitro. Cyclic beef cows in Treatment A and pregnant cows in Treatment B were administered a total dose of 40 mg of FSH in descending dose levels (6, 5, 4, 3 and 2 mg) twice daily for 5 consecutive days. Cows in Treatment A were administered 25 mg of PGF(2)alpha and in Treatment B an equal volume of 0.9% saline at the seventh FSH injection. Pregnant cows in Treatment C were administered neither FSH nor PGF(2)alpha and served as a control group. Following a gonadotropin treatment, the ovaries of each female were evaluated for follicular development by ultrasonography. Oocytes were collected by follicle aspiration from cows in the first trimester. Following IVF procedures, the embryos were co-cultured on caprine oviductal cells, or in the chicken embryo co-culture system, or were placed in goat oviducts in vivo. The mean number of follicles per ovary 12 hours after FSH treatment was not different for cows in Treatments A and B, (8.1 vs 7.7) and both numbers were greater (P<0.05) than the 1.1 follicles per ovary for the control cows in Treatment C. Oocytes collected in vivo and exposed to IVF, resulted in 20% cleaving, and of these embryos 50% developed to the morula stage in culture. In summary, stimulating supplemental follicular development with FSH treatment during pregnancy and collecting the oocytes for IVF may be an alternative method for obtaining supplemental gametes from valuable donor cattle.

Journal Article↗
PubMed1993

Mutational specificity of the (+)-anti-diol epoxide of benzo[a]pyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancement of mutagenesis.

Mutagenesis by the suspected major mutagenic metabolite of activated benzo[a]pyrene, which is (+)-anti-BPDE, was analyzed with a new system, which permits the selection of supF- mutations in an Escherichia coli plasmid using lactose minimal plates. (+)-anti-BPDE enhances base pairing mutations--principally at G:C base pairs, frameshift mutations and large deletions. Frameshift mutagenesis principally involves deletions and insertions of a single G:C base pair in runs of G:C base pairs. Base pairing mutations are significantly enhanced by SOS induction, especially GC-->TA mutations. Nearest neighbor analysis was performed assuming that a guanine (underlined) is being mutated, and (+)-anti-BPDE base pairing mutagenesis is enhanced by SOS induction in 5'-(A/T)G-3' sequences approximately 4-fold more than in 5'-(G/C)G-3' sequences, and in 5'-G(C/G)-3' sequences approximately 4-fold more than in 5'-G(A/T)-3' sequences; this is discussed. The influence of sequence context on quantitative aspects of (+)-anti-BPDE mutagenesis is considered, and hotspots are found at most, but not all, 5'-GG-3' sequences. The influence of sequence context on qualitative aspects of (+)-anti-BPDE mutagenesis (i.e. mutagenic specificity) is also considered. For example, the sequences, 5'-AG-3', 5'-CG-3' and 5'-GG-3', all have examples of G-->T, G-->A and G-->C mutations, while in the sequence, 5'-TG-3', only G-->T mutations have been detected. (The latter finding correlates with a recent site-specific study on the major adduct of (+)-anti-BPDE formed at N2-Gua in a 5'-TG-3' context, where G-->T mutations predominated [Carcinogenesis (1992) 13, 1415-1425].) These results suggest that sequence context plays a role in defining the kind of mutation (i.e. GC-->TA versus GC-->AT versus GC-->CG) induced by (+)-anti-BPDE, where the base on the 5' side of the guanine undergoing mutation seems to be influential. The most likely model for this is that sequence context influences adduct conformation, which controls mutagenic specificity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗