Post-menopausal bleeding - Is transvaginal ultrasound a useful first-line investigation in tamoxifen users?

Objective To evaluate the role of transvaginal ultrasound triage in women with a history of tamoxifen treatment who present with post-menopausal bleeding. Study design A retrospective review was undertaken of patients who presented with symptoms of post-menopausal bleeding and underwent ultrasound triage. Endometrial thickness and ultrasonographic features were then correlated with hysteroscopic and histopathological outcome data. The findings and outcomes for women with a history of tamoxifen use (tamoxifen group) were compared to those who had not taken tamoxifen (non-tamoxifen group). Results A total of 614 women with post-menopausal bleeding underwent transvaginal ultrasound triage, of whom 53 had a history of current or previous tamoxifen treatment. An endometrial thickness of ≥5 mm or the presence of other abnormal features was used to triage women to further investigation by hysteroscopy and biopsy. Endometrial thickness was significantly greater in the tamoxifen group (11 mm vs. 6 mm). Nearly all of the tamoxifen group were triaged to further investigation (98.1%), compared with significantly fewer in the non-tamoxifen group (68.3%) Overall, the incidence of endometrial pathology was also significantly higher in tamoxifen patients (43.4% vs. 31.7%). Conclusion For women presenting with post-menopausal bleeding, the use of transvaginal ultrasound as a triage tool is rarely helpful in evaluating women who have a history as tamoxifen use, as most will require further investigation with hysteroscopy and biopsy. A post-menopausal bleeding protocol that omits transvaginal ultrasound for women with a history of tamoxifen use may be an appropriate and effective pathway for managing these patients.

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1.

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1.

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

Incidence and demographic correlates of eating disorder symptoms in a pregnant population.

OBJECTIVE: To obtain a picture of eating disorder symptoms in a population of pregnant women. METHOD: Five hundred thirty women attending antenatal follow-up clinics at a large London district general hospital during a 4-week period were surveyed. The Eating Attitudes Test (EAT), the Edinburgh Postnatal Depression Scale (EPDS), and a demographic questionnaire were administered. Unadjusted relative risks and their 95% confidence intervals were calculated for a series of prognostic factors. RESULTS: 4.9% of women scored above the recommended threshold on the EAT in pregnancy. Eating disorder symptomatology was found to be associated with younger age, previous symptomatology, lower educational attainment, poorer housing, employment status, and previous miscarriage. DISCUSSION: The meaning and potential implications of high levels of eating disorder symptomatology in a pregnant population are discussed in the light of the physiological and psychological effects of eating disorders on both pregnancy outcome and infant development.

Purification and characterization of EDTA monooxygenase from the EDTA-degrading bacterium BNC1.

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35 degreesC. Kms were 34.1 microM for uncomplexed EDTA and 8.5 microM for MgEDTA2-; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2-. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.

Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR.

Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 10(5) to 10(6) copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 microgram of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 10(5) to 10(6) copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 10(5) to 10(6) copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.

Incidence and demographic correlates of depressive symptoms during pregnancy in an inner London population.

This paper reviews the literature on antenatal depression and reports on a recent survey of depressive symptoms among 407 women attending an inner London antenatal clinic. The high level of depressive symptoms in this survey was in line with that recently reported from a deprived inner-city population in the United States. Depressive symptoms were associated with having no educational qualifications, being unmarried, the woman being unemployed, having poor support from a partner if present, and being in second or subsequent pregnancy. Doctors should be aware that women are not especially protected from symptoms of depression during pregnancy. The association with socioeconomic problems suggests that this is one aspect of morbidity among disadvantaged people. There is a need for more research on the causes of depression during pregnancy and on evaluation of medical treatment and social intervention.

Effects of a lignin peroxidase-expressing recombinant, Streptomyces lividans TK23.1, on biogeochemical cycling and the numbers and activities of microorganisms in soil.

A recombinant actinomycete, Streptomyces lividans TK23.1, expressing a pIJ702-encoded extracellular lignin peroxidase gene cloned from the chromosome of Streptomyces viridosporus T7A, was released into soil in flask- and microcosm-scale studies to determine its effects on humification and elemental cycling and on the numbers, types, and activities of microorganisms native to the soil. Strain TK23.1 had been shown previously to transiently increase the rate of organic carbon mineralization in soil via an effect that was recombinant specific and particularly significant in nonsterile soils already possessing an active microflora. The results of this study confirmed the previous findings and showed that additional effects were measurable upon release of the recombinant strain TK23.1 into unamended soil and into soil amended with lignocellulose. In addition to a transient enhancement of carbon mineralization, the recombinant affected soil pH, the rate of incorporation of carbon into soil humus fractions, nitrogen cycling, the relative populations of some microbial groups, and also certain soil enzyme activities. Whereas the survival or persistence in soil of the recombinant TK23.1 strain and that of its parent, TK23, were similar, the observed effects on microbial numbers, types, and activities were recombinant specific and did not occur when the parental strain was released into soil. All of the measured effects were transient, generally lasting for only a few days. While the effects were statistically significant, their ecological significance appears to be minimal. This is the first report showing that a recombinant actinomycete can affect the microbial ecology of soil in ways that can be readily monitored by using a battery of microbiological, enzymological, and chemical assays.

Field calibration of soil-core microcosms: Fate of a genetically altered rhizobacterium.

Microcosms containing intact soil-cores are a potential tool for assessing the risks of the release of genetically engineered microorganisms (GEMs) to the environment. Before microcosms become a standard assessment tool, however, they must first be calibrated to ensure that they adequately simulate key parameters in the field. Four systems were compared: intact soil-core microcosms located in the laboratory at ambient temperature and in a growth chamber with temperature fluctuations that simulated average conditions in the field, field lysimeters, and field plots. These four systems were inoculated with rifampicin-resistantPseudomonas sp. and planted to winter wheat. Populations of thePseudomonas sp. in soil decreased more rapidly at ambient temperature, but population size at the three-leaf stage of wheat growth was the same in all four systems. Populations of thePseudomonas sp. on the rhizoplane of wheat were the same at the three-leaf stage in all four systems, and colonization with depth at the final boot stage-sampling was also similar. In general, microcosms incubated at ambient temperature in the laboratory or in the growth chamber were similar to those in the field with respect to survival of and colonization of the rhizoplane by the introducedPseudomonas sp.

Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons.

Microcosms containing intact soil-cores are a potential biotechnology risk assessment tool for assessing the ecological effects of genetically engineered microorganisms before they are released to the field; however, microcosms must first be calibrated to ensure that they adequately simulate key field parameters. Soil-core microcosms were compared with the field in terms of ecological response to the introduction of a large inoculum of a rifampicin-resistant rhizobacterium,Pseudomonas sp. RC1. RC1 was inoculated into intact soil-core microcosms incubated in the laboratory at ambient temperature (22°C) and in a growth chamber with temperature fluctuations that mimicked a verage field values, as well as into field lysimeters and plots. The effect of the introduced bacterium on ecosystem structure, including wheat rhizoplane populations of total and fluorescent pseudomonads, total heterotrophic bacteria, and the diversity of total heterotrophic bacteria, was determined. Fluorescent pseudomonads were present on the rhizoplane in significantly lower numbers in soil inoculated with RC1, in both microcosms and the field. Conditions for microbial growth appeared to be most favorable in the growth chamber microcosm, as evidenced by higher populations of heterotrophs and a greater species diversity on the rhizoplane at the three-leaf stage of wheat growth. Ecosystem functional parameters, as determined by soil dehydrogenase activity, plant biomass production, and(15)N-fertilizer uptake by wheat, were different in the four systems. The stimulation of soil dehydrogenase activity by the addition of alfalfa was greater in the microcosms than in the field. In general, growth chamber microcosms, which simulated average field temperatures, were better predictors of field behavior than microcosms incubated continuously at 22°C.

Human lactase and the molecular basis of lactase persistence.

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in Mr (160K). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl-beta-galactoside and beta-glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with lactase under all conditions investigated; coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.