Advances in cereal functional genomics.
Explore the source record for details and available documents.
Arabidopsis↗
Biomedical subjects
R Chibbar
Publications and source records attributed to R Chibbar.
bcl-1 gene rearrangements in mantle cell lymphoma: a comprehensive analysis of 118 cases, including B-5-fixed tissue, by polymerase chain reaction and Southern transfer analysis.
We evaluated 118 cases of mantle cell lymphoma by polymerase chain reaction (PCR) for the major translocation cluster (MTC) region and another breakpoint corresponding to probe p94PS, located 24 kb telomeric to the MTC locus on chromosome 11. The specimens included 64 frozen, 19 formalin-fixed, and 9 B-5-fixed lymph nodes and 26 B-5-fixed bone marrow biopsy specimens. We also analyzed DNA from the 64 frozen lymph nodes by Southern transfer analysis (SB) using three separate bcl-1 breakpoint probes. Gene rearrangements were identified in 17 (PCR) and 18 (SB) of 64 frozen lymph nodes and by PCR in 6 of 19 formalin-fixed lymph nodes, 3 of 9 B-5-fixed lymph nodes, and 12 of 26 B-5-fixed bone marrow cores with MTC locus primers and probe. Only one case showed rearrangement with the p11EH probe that corresponds to breakpoints situated 63 kb telomeric to the MTC locus. No rearrangements were detected by PCR or SB for the breakpoint site corresponding to the p94PS probe, but we identified a polymorphic restriction site with HinD III digest in approximately 25% of the cases. In agreement with other studies, these results confirmed that breakpoints in the MTC region of the bcl-1 locus are tightly clustered and associated with 30 to 40% of mantle cell lymphomas. Other breakpoints in the bcl-1 locus seem to be heterogeneous and cannot be detected by PCR or SB with use of existing probes or primer sequences. The most important finding of our study is optimization of the methodology for the detection of immunoglobulin heavy chain gene rearrangement and MTC region breakpoints by PCR from the DNA isolated from B-5-fixed, paraffin-embedded lymph nodes and bone marrow biopsy specimens. The results obtained from these tissues are comparable to those obtained from frozen or formalin-fixed tissue.
Blotting, Southern↗
Detection of the factor VLeiden mutation. Development of a testing algorithm combining a coagulation assay and molecular diagnosis.
The Coagulation and Molecular Diagnostic laboratories at the University of Minnesota Medical School (Minneapolis) have collaborated to develop a diagnostic algorithm to identify all factor VLeiden mutation carriers without performing unnecessary and expensive genetic testing. The algorithm uses a coagulation assay for activated protein C resistance (APCR) to determine the need for genetic testing. We report the results of our experience validating this program. We compared the sensitivity, specificity, and positive and negative predictive values of two measures of APCR, the APCR ratio and the normalized ratio. We found that the normalized ratio was the more sensitive but less specific parameter to determine the need for genetic testing. By using the normalized ratio as the standard by which to refer patients to the Molecular Diagnostics Laboratory, all mutation carriers were identified. We found a large overlap in both measures of APCR between symptomatic patients with normal genotype and mutation carriers. Furthermore, we demonstrated that increased factor VIII levels with a normal genotype are associated with apparent APCR. In this article we also review other correlates of apparent APCR.
Adult↗
Correlation of PCR-detected clonal gene rearrangements with bone marrow morphology in patients with B-lineage lymphomas.
Bone marrow biopsy is the conventional staging and posttherapy evaluation method for assessing marrow involvement by lymphoma. Polymerase chain reactions (PCR) for antigen receptor rearrangements have the potential to increase the detection of minimal degrees of marrow involvement. The present study is a concurrent morphologic and PCR evaluation of 225 staging or posttherapy marrow biopsies from 127 patients with B-lineage non-Hodgkin's lymphoma. The biopsies were morphologically categorized into four groups: group 1 (positive for lymphoma), 60 biopsies (27%); group 2 (suspicious for lymphoma), 20 biopsies (9%); group 3 (lymphocytic lesions of indeterminate biology), 22 biopsies (10%); and group 4 (negative for lymphoma), 123 biopsies (54%). Molecular studies were performed on concurrently obtained aspirates and used consensus immunoglobulin-heavy-chain (IgH) and IgH/bcl-2 gene PCR primers. A molecular clone was detected in 53 of the 225 aspirates (24%): group 1, 34 aspirates (57%); group 2, five aspirates (25%); group 3, one aspirate (5%); and group 4, 13 aspirates (11%). A PCR-positive aspirate was present in 47% of follicular lymphomas, 58% of diffuse large cell lymphomas, and 72% of the other lymphomas in the group I specimens. Morphology or PCR was positive in 79 of the 225 cases (35%). The molecular detection of clonality in the aspirate DNA from cases with positive morphologic findings was lower than anticipated. The discordance between morphology and PCR results may be related to sample variation between the trephine biopsy and aspirate, a failure to aspirate sufficient lymphoma cells, or insufficient primer homology for amplification. DNA extracted from trephine sections may provide results more concordant with morphology, because PCR detected a clone in 10 of 11 DNA specimens extracted from trephine biopsies with positive morphologic findings and PCR negative aspirates.
Base Sequence↗
Estrogen stimulates oxytocin gene expression in human chorio-decidua.
We have recently shown that oxytocin (OT) is synthesized within human amnion, chorion, and decidua during late gestation. The levels of OT messenger ribonucleic acid (mRNA) increased around the time of parturition, suggesting that locally produced OT may play a role in this poorly understood process. In this report, we present results from investigations into the effects of estrogen and progesterone on the synthesis of OT by human chorio-decidua. Using an in vitro incubation system, estradiol at physiological concentrations more than doubled the concentration of OT mRNA. This was reflected by an increase in the amount of OT peptide secreted into the medium. The increase in OT mRNA was antagonized by tamoxifen, suggesting that the effects were estrogen receptor mediated. Progesterone had no effect on OT mRNA synthesis. Using ribonuclease protection assays, mRNAs for estrogen receptor (ER) and progesterone receptor (PR) were detected in all tissues examined. The highest levels were found in decidua, with lower amounts in chorion and very small amounts in amnion and placenta. This is the same relative tissue distribution that we previously demonstrated for OT mRNA. A single transcript was present for ER, and two transcripts were protected for PR. The concentrations of ER mRNA in chorio-decidua were 3-fold higher in tissues obtained after spontaneous labor onset than in tissues obtained from cesarean section at a similar gestational age but before labor onset. Levels of PR did not change significantly. We conclude that synthesis of OT in human chorio-decidua may be regulated in part by estrogen, and that regulation of ER levels may be an important factor modulating this effect. These data support the hypothesis of a paracrine network within human fetal membranes and decidua that may participate in regulating the timing of human birth.
Chorion↗
Synthesis and metabolism of oxytocin in late gestation in human decidua.
Several studies in the past few years have supported the hypothesis that oxytocin (OT) is synthesized in a paracrine system within the pregnant human uterus and that this paracrine system may be an important regulator of the timing of human parturition. Using ribonuclease protection assays, we have demonstrated a three-fold increase in the rate of synthesis of OT mRNA in human decidua around the time of parturition. We also have shown that a similar increase in OT mRNA and peptide synthesis can be stimulated in vitro by physiological concentrations of estradiol. This increase is inhibited by concomitant use of the estrogen receptor (ER) blocker tamoxifen or by transcription inhibitors. Progesterone had little, if any effect. We also detected mRNAs for ER and progesterone receptor (PR) in amnion, chorion and decidua with the same relative tissue concentrations as OT mRNA. The concentrations of ER but not PR increased significantly around the time of labour onset. To determine if local OT concentrations may be regulated by changes in OT metabolism, we determined kinetic parameters for OT metabolism in decidua, chorion and placenta. [3H]tyrosyl-OT was used as substrate. Metabolites were separated using HPLC and identified using amino acid analysis and mass spectrometry. Metabolism in decidua and chorion occurred predominantly via a cytosolic post-proline endopeptidase and the activity was comparable to placenta. In microsomal fractions, cystine aminopeptidase activity predominated and placenta had significantly more activity than decidua and chorion. There were no changes in any Km or apparent vmax values around the time of parturition. These findings support the existence of a paracrine system within human decidua that involves sex steroids regulating synthesis of OT and that undergoes significant changes around the time of parturition. Changes in local OT concentrations are controlled by rates of synthesis rather than rates of metabolism.
Amino Acid Sequence↗
Synthesis of oxytocin in amnion, chorion and decidua: a potential paracrine role for oxytocin in the onset of human parturition.
Explore the source record for details and available documents.
Amnion↗
Synthesis of oxytocin in amnion, chorion, and decidua may influence the timing of human parturition.
Despite the widespread clinical use of oxytocin (OT) as a potent and specific stimulant of labor, previous research data have not supported a role for OT in the physiology of normal human parturition. We have demonstrated synthesis of OT mRNA in amnion, chorion, and decidua using Northern blot analysis, ribonuclease protection assays, and in situ hybridization. Probes directed towards both the 3' and 5' ends of the gene have been used. Levels were highest in decidua with considerably less in chorion and amnion and very low levels in placenta. The transcript size in decidua appears to be 60-80 nucleotides smaller than the transcripts in amnion and chorion. OT gene expression in chorio-decidual tissues increased three- to fourfold around the time of labor onset. Estradiol stimulated synthesis of OT mRNA during in vitro incubation. These results support the hypothesis of a paracrine system involving OT and sex steroids within intrauterine tissues wherein significant changes could occur without being reflected in the maternal circulation. Such a paracrine system could rationalize a long-sought role for oxytocin in the physiology of human labor. These data may lead to novel approaches towards prevention or treatment or preterm labor.
Amnion↗
Steroid sulfohydrolase in human chorion and decidua: studies using pregnenolone sulfate and dehydroepiandrosterone sulfate as substrate.
Human chorion and decidua use pregnenolone sulfate (P5S) and dehydroepiandrosterone sulfate (DHAS) as substrates for local estrogen and progesterone synthesis. We hypothesized that the local estrogen/progesterone ratio may influence contractility of the adjacent myometrium and hence effect the timing of parturition. Thus, we studied steroid sulfohydrolase activity for P5S in these tissues and investigated the potential interaction of other steroids on the rates of hydrolysis of P5S and DHAS. The enzyme was present in both tissues, predominantly in the microsomal fraction. With P5S as substrate, the Michaelis-Menten constant (Km) was similar in chorion (1.3 +/- 0.2 mumol/L, mean +/- SEM) and decidua (0.9 +/- 0.1 mumol/L) but the maximum velocity (Vmax) was significantly greater in chorion (2.6 +/- 0.4 vs. 1.1 +/- 0.3 nmol/mg protein/15 min, P less than 0.05). In both tissues there was a tendency towards greater activity in tissues obtained before labor compared to tissues obtained after spontaneous labor onset. Using either DHAS or P5S as substrate, there was significant inhibition of sulfohydrolase activity by other steroids at concentrations similar to those in late pregnancy fetal and maternal plasma. In microsomal preparations using DHAS as substrate, activity was inhibited by equimolar concentrations of estrone sulfate (E1S, by 38 +/- 2%), P5S (by 74 +/- 2%), and cholesterol sulfate (C27S, by 38 +/- 3%). With P5S as substrate, equimolar concentrations of E1S, DHAS, and C27S caused inhibition of sulfohydrolase activity by 19 +/- 5%, 16 +/- 4%, and 18 +/- 2%, respectively. These inhibitory effects also were observed using a tissue explant system with intact cells. In kinetic inhibition studies using DHAS as substrate, E1S and P5S were competitive inhibitors with inhibition constants (Ki) of 4.8 +/- 1.3 and 0.7 +/- 0.1 mumol/L, respectively. Using P5S as substrate, E1S and DHAS also were competitive inhibitors with Ki values of 8.2 +/- 2.1 and 9.6 +/- 1.2 mumol/L, respectively. For both substrates, the pattern of inhibition by C27S was complex. Preliminary experiments to distinguish, on the basis of differing physical-chemical properties, separate enzymes for different substrates were inconclusive. We conclude that human chorion and decidua can hydrolyze several steroid sulfoconjugates and this activity may regulate local estrogen and progesterone synthesis. There are significant interactions among steroid sulfoconjugates in regulating this activity. These activities may be important components of a paracrine system that determines myometrial contractility and the timing of parturition.
Arylsulfatases↗
Regulation of neural oxytocin gene expression by gonadal steroids in pubertal rats.
We have previously demonstrated that neuronal oxytocin mRNA increases during the pubertal development of female rats. In this paper we have examined the factors that regulate this developmental increase in both male and female rats. Northern blot analysis demonstrated that neural oxytocin mRNA increased 5- to 10-fold from postnatal day 20 (P20) to P60 in animals of both sexes, coincident with puberty. Mature male rats and females at all stages of the estrous cycle expressed similar levels of neural oxytocin mRNA. Pubertal up-regulation of oxytocin mRNA was largely, but not completely, inhibited by prepubescent gonadectomy, indicating a requirement for intact gonads as well as some other as yet undefined factor(s). Pubertal treatment of gonadectomized animals with estradiol or testosterone abolished the effects of gonadectomy; treated animals expressed levels of neural oxytocin mRNA similar to those in controls. However, treatment of prepubertal animals with estradiol or testosterone from P10 to P20 had no effect on oxytocin mRNA levels, suggesting that neural maturation or other factors are necessary requisites for steroid sensitivity. To determine whether neural activin played any role in regulating oxytocin mRNA during puberty, we examined levels of inhibin/activin beta A-chain mRNA. This mRNA was expressed at similar levels in all brain regions and did not vary as a function of gonadectomy or steroid treatment, making it unlikely that activin mediates the observed changes. Together, these data indicate that neural oxytocin mRNA is induced by gonadal steroids during puberty, and suggest a mechanism for coordinating development of reproductive functions with other pubertal changes.
Activins↗
Steroid sulfohydrolase activity in human chorion. I. Interactions of other steroids with estrone sulfate as substrate.
Human chorion contains steroid sulfohydrolase activity and synthesizes free estrogens from estrone sulfate (E1S). We hypothesized that the free estrogen thus formed may influence the contractility of the adjacent myometrium in late pregnancy. In this study we measured the abilities of various steroids and steroid conjugates to influence the hydrolysis of E1S by the 105,000 x g pellet of chorion tissue obtained from women after spontaneous onset of labor and vaginal delivery and from women delivered by cesarean section before labor onset. No differences were found in tissues obtained before or after the onset of labor. None of the steroids increased the rate of hydrolysis. Several unconjugated steroids caused significant inhibition, but only at concentrations well beyond physiological ranges in maternal or fetal blood. However, conjugated steroids had marked inhibitory effects at circulating concentrations. At equimolar concentrations with the E1S substrate, the sulfoconjugates of dehydroepiandrosterone, pregnenolone, and cholesterol caused 27 +/- 1% (+/- SE), 64 +/- 1%, and 40 +/- 1% inhibition, respectively. These results were confirmed using a tissue explant system. Using enzyme inhibition kinetic analysis, we determined that the inhibition by dehydroepiandrosterone sulfate was non-competitive, with Ki = 8.7 +/- 1.7 mumol/L. The inhibition by pregnenolone sulfate was competitive, with Ki = 1.6 +/- 0.4 mumol/L, and that by cholesterol sulfate was primarily noncompetitive, with Ki = 7.4 +/- 1.2 mumol/L. We conclude that there is significant interaction among sulfurylated steroids that may influence local free estrogen synthesis within human chorion. This interaction may affect the contractility of the late pregnancy myometrium.
Chorion↗
Sulfohydrolase activity for estrone sulfate and dehydroepiandrosterone sulfate in human fetal membranes and decidua around the time of parturition.
We examined the distribution and kinetic parameters of sulfohydrolase activity in human amnion, chorion, and decidua using estrone sulfate (E1S) and dehydroepiandrosterone sulfate as substrates. Amnion contained low levels of sulfatase activity. Chorion had active sulfohydrolase activity for both substrates, but a significantly greater maximum velocity (Vmax) for E1S. The Km was not different between the two substrates. However, there was a slight but statistically significant decrease in Km and increase in Vmax for sulfohydrolase activity using E1S in chorion from patients delivering vaginally after the spontaneous onset of labor compared to those delivering by elective cesarean section before the onset of labor but at a similar gestational age. Decidua possessed sulfohydrolase for E1S with similar Km and Vmax as chorion. There were no changes occurring around the onset of labor. Using dehydroepiandrosterone sulfate as substrate, the decidua had a similar Km as the chorion, but its Vmax was significantly less. In both tissues for both substrates, the enzyme had highest specific activity in the 105,000 X g pellet, with almost no activity in the soluble fraction. The greatest total sulfohydrolase activity was contained in the 800 X g pellet despite several methods of homogenization and washing of the 800 X g pellet. We conclude that the sulfohydrolase activity of human chorion and decidua may be an important factor in regulating free steroid levels within the pregnant uterus. The significant change in the kinetic parameters of E1S sulfatase may partially explain the increased ability of chorion to hydrolyze E1S which occurs in association with the spontaneous onset of labor.
Carbon Radioisotopes↗