A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy.

OBJECTIVE: UK colposcopy services are seeing increased workloads, a large proportion of which are follow-up appointments. The English Cervical Screening Programme HPV Special Interest Group identified five subcategories of colposcopy clinic patients who often require prolonged follow-up regimes for low-grade abnormalities. Human papillomavirus (HPV) testing has a high negative predictive value, meaning that HPV-negative women are at very low risk of underlying disease. Our objectives were to quantify the number of HPV-negative women in each study subcategory and to evaluate the number who could potentially be discharged from colposcopy on the basis of their results. METHODS: Four colposcopy clinics prospectively identified women according to five categories over 12 months. All women underwent cytological testing and high-risk HPV (hrHPV) testing using the Hybrid Capture 2 test. Management outcomes and decisions based on a knowledge of the HPV status were recorded. RESULTS: Data available on 755 women showed that 422/755 (55.9%) and 260/755 (34.4%) had persistent cervical intraepithelial neoplasia grade 1 (CIN1) (Category 1) or a minor abnormality following treatment (Category 2), respectively. In Categories 1 and 2, 51.7% and 60.2%, respectively, were hrHPV negative. The rates with biopsies of CIN2 or worse (CIN2+) across the two categories were 3/355 (0.8%) and 21/291 (7.0%) for hrHPV-negative and hrHPV-positive women, respectively. CONCLUSION: The incorporation of hrHPV testing within organized cervical screening programmes has been widely accepted. hrHPV testing for the clinical scenarios outlined in this study detects women who are hrHPV negative and therefore at low risk of underlying disease, potentially reducing anxiety and inconvenience for women and costs to colposcopy services.

Guidelines for the management of vulvodynia.

These guidelines for the management of vulvodynia have been prepared by the British Society for the Study of Vulval Diseases Guideline Group. They present evidence-based guidance for treatment, with identification of the strength of evidence available at the time of preparation of the guidelines.

Uterine tumour resembling an ovarian sex cord tumour.

Endometrial stromal sarcomas account for 0.25% of all uterine malignancies. These tumours were originally divided into low grade and high grade stromal sarcomas, but the recent World Health Organisation classification (2003) recognises low grade stromal sarcoma and undifferentiated endometrial sarcoma. Low grade sarcomas may exhibit other forms of differentiation, including smooth muscle and sex cord differentiation. In the latter form, the tumour contains epithelial-like or sex cord-like elements often with epithelioid appearance, arranged in nests, cords, trabeculae, solid, or tubular structures. If this element predominates, the tumour is considered to be a uterine tumour resembling ovarian sex cord tumour, and may cause diagnostic difficulties. This case report describes the histological and immunohistochemical features of a uterine stromal sarcoma showing exclusively a pattern reminiscent of ovarian sex cord tumour.

Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development.

Medicago truncatula contains a family of at least five genes related to AUX1 of Arabidopsis thaliana (termed MtLAX genes for Medicago truncatula-like AUX1 genes). The high sequence similarity between the encoded proteins and AUX1 implies that the MtLAX genes encode auxin import carriers. The MtLAX genes are expressed in roots and other organs, suggesting that they play pleiotropic roles related to auxin uptake. In primary roots, the MtLAX genes are expressed preferentially in the root tips, particularly in the provascular bundles and root caps. During lateral root and nodule development, the genes are expressed in the primordia, particularly in cells that were probably derived from the pericycle. At slightly later stages, the genes are expressed in the regions of the developing organs where the vasculature arises (central position for lateral roots and peripheral region for nodules). These results are consistent with MtLAX being involved in local auxin transport and suggest that auxin is required at two common stages of lateral root and nodule development: development of the primordia and differentiation of the vasculature.

Perception of lipo-chitooligosaccharidic Nod factors in legumes.

Lipo-chitooligosaccharides produced by rhizobia are a class of signalling molecules that mediate recognition and nodule organogenesis in the legume-rhizobia symbiosis. Their synthesis is specified by the nodulation genes of rhizobia and hence they are commonly known as Nod factors. They are amphiphilic molecules and induce a variety of responses in the roots of the legume hosts. Studies using plant and rhizobial mutants and purified molecules suggest that Nod factors are recognized by more than one receptor. In this article, we review evidence about the affinity, specificity and location of these putative receptors and describe recent studies with regard to their identification.

Differential expression of the two cytosolic glutamine synthetase genes in various organs of Medicago truncatula.

In order to clarify the physiological roles of the cytosolic forms of glutamine synthetase (GS) in Medicago truncatula, we have performed a detailed analysis of the expression of the two functional cytosolic GS genes, MtGSa and MtGSb in several organs of the plant. Transcriptional fusions were made between the 2.6 or 3.1 kbp 5' upstream regions of MtGSa or MtGSb, respectively, and the reporter gene gusA encoding beta-glucuronidase and introduced into the homologous transgenic system. MtGSa and MtGSb were found to be differentially expressed in most of the organs, both temporally and spatially. The presence of GS proteins at the sites where the promoters were active was confirmed by immunocytochemistry, providing the means to correlate gene expression with the protein products. These studies have shown that the putative MtGSa and MtGSb promoter fragments were sufficient to drive GUS expression in all the tissues and cell types where cytosolic GS proteins were located. This result indicates that the cis acting regulatory elements responsible for conferring the contrasting expression patterns are located within the region upstream of the coding sequences. MtGSa was preferentially expressed in the vascular tissues of almost all the organs examined, whereas MtGSb was preferentially expressed in the root cortex and in leaf pulvini. The location and high abundance of GS in the vascular tissues of almost all the organs analysed suggest that the enzyme encoded by MtGSa plays an important role in the production of nitrogen transport compounds. The enzyme synthesised by MtGSb appears to have more ubiquitous functions for ammonium assimilation and detoxification in a variety of organs.

Cellular expression and regulation of the Medicago truncatula cytosolic glutamine synthetase genes in root nodules.

In this paper we have studied the localisation of expression of the two functional cytosolic glutamine synthetase (GS) genes, MtGSa and MtGSb, in root nodules of the model legume Medicago truncatula. We have used a combination of different techniques, including immunocytochemistry, in situ hybridisation and promoter beta-glucuronidase (GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation. These studies revealed that transcriptional regulation (mRNA synthesis) plays an important part in controlling GS protein levels in nodules of M. truncatula. The major locations of cytosolic GS mRNA and protein are the central tissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GS might be involved in other physiological processes in addition to the primary assimilation of ammonia released by the bacterial nitrogenase. The two genes show different but overlapping patterns of expression with MtGSa being the major gene expressed in the infected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb of MtGSa and MtGSb, respectively, have been sequenced and primer extension revealed that the MtGSb promoter is expressed in nodules from an additional start site that is not used in roots. Generally these fragments in the homologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell types where GS proteins and transcripts are located except that the MtGSa promoter fragment did not express GUS highly in the nodule infected cells. These results indicate that the cis-acting regulatory elements responsible for infected-cell expression are missing from the MtGSa promoter fragment.

The presence of GSI-like genes in higher plants: support for the paralogous evolution of GSI and GSII genes.

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution.

The abnormal glandular smear: cytologic prediction, colposcopic correlation and clinical management.

We reviewed recent cytological reporting of abnormal glandular cells on cervical smears in order to assess the predictive value of these reports and the contribution of colposcopy in the assessment of these abnormalities. The study consisted of a 5-year retrospective review of the clinical management of 80 women with abnormal glandular cells on a cervical smear, with clinical and histopathological data available for review in the interval 1992-1996. There were two groups of women: (i) those referred with gynaecological symptoms and (ii) those with screen detected abnormalities who were asymptomatic and significantly younger than the first group. The predictive value of a glandular smear for malignancy was 42.5% and for premalignancy 28.8%. The most common lesions detected were cervical intraepithelial neoplasia (CIN) (13), endometrial cancer (13), cervical adenocarcinoma (10) and cervical intraepithelial glandular neoplasia (CIGN) (8). Four cases of endometrial carcinoma presented through screening. In the remainder a variety of benign conditions were identified as responsible for the abnormal smear. Failure to find an explanation for the abnormal smear only occurred in 8.8%. In developing a protocol for abnormal glandular smears, our observations indicate that: (a) those with abnormal bleeding require endometrial sampling; (b) for those with screen detected abnormality, colposcopy is valuable as it is a sensitive predictor of early invasion and can predict glandular abnormality; (c)diseases of the entire genital tract, non-gynaecological viscera and metastatic cancer can generate cytological abnormality; (d) screen detected borderline abnormality in endocervical cells is associated with CIN III.

Recent advances in the study of nod factor perception and signal transduction.

Rhizobial lipochitooligosaccharidic Nod factors mediate the specific recognition between leguminous plants and their prokaryotic symbionts. This review summarizes recent findings on the way plants could perceive and transduce these bacterial signals. It starts by summarizing knowledge about Nod factor binding sites, before moving to the potential implications in Nod factor signal transduction of G proteins, root-hair plasma membrane depolarisation, cytoplasmic and extracellular alkalinisation and finally variations in cytoplasmic calcium concentration.

Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures.

Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.

The Medicago truncatula MtAnn1 gene encoding an annexin is induced by Nod factors and during the symbiotic interaction with Rhizobium meliloti.

Here we report the characterization of a new Nod factor-induced gene from Medicago truncatula identified by mRNA differential display. This gene, designated MtAnn1, encodes a protein homologous to the annexin family of calcium- and phospholipid-binding proteins. We further show that the MtAnn1 gene is also induced during symbiotic associations with Rhizobium meliloti, both at early stages in bacterial-inoculated roots and in nodule structures. By in situ hybridization, we demonstrate that MtAnn1 expression in nodules is mainly associated with the distal region of invasion zone II not containing infection threads, revealing MtAnn1 as a new marker gene of the pre-infection zone. Moreover, analyses of MtAnn1 expression in response to bacterial symbiotic mutants suggest that the expression of MtAnn1 during nodulation requires biologically active Nod factors and is independent of the infection process.

Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme.

We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.

Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development.

We report the identification of new molecular markers associated with different stages of Rhizobium-induced nodule development in the legume Medicago truncatula. A cDNA library was constructed from pre-nitrogen-fixing M. Truncatula nodules, and differentially screened with a polymerase chain reaction-amplified subtracted probe. Twenty-nine new families of nodulin cDNA clones, designated MtN1 to MtN29, were thus identified in addition to clones for several known nodulins. All MtN genes were shown by Northern (RNA) hybridization analysis to be induced during nodulation, some of them well before nodule emergence. The MtN genes were classified into three groups depending on their expression kinetics. The expression of three MtN genes showed a limited induction by Nod factors purified from Rhizobium meliloti. Homologies with a variety of proteins were found for the deduced amino acid sequences of 10 of the MtN genes.

Characterization of a binding site for chemically synthesized lipo-oligosaccharidic NodRm factors in particulate fractions prepared from roots.

This paper describes the characteristics of a binding site for the major, lipo-oligosaccharide Nod factor of Rhizobium meliloti in roots of the symbiotic host plant, Medicago truncatula. Chemically synthesized NodRm-IV(Ac, S, C16:2) was labelled by tritiation to a specific activity of 56 Ci mmol-1 and this ligand was shown to be biologically active in the root hair deformation assay at 10(-11) M. Binding of the ligand to a particulate fraction from roots of M. truncatula was found to be saturable and reversible with an affinity (Kd) of 86 nM and the binding characteristics were consistent with a single class of binding sites. Competition with modified Nod factors showed that the binding was independent of both the O-acetyl and the sulphyl group and did not depend on the unsaturation of the fatty acid. However, both moieties of the lipo-oligosaccharide are required for high-affinity binding since tetra-N-acetyl-chitotetraose and palmitate were found to be poor competitors of ligand binding. A binding site with analogous characteristics was also found in a similarly prepared particulate fraction of tomato roots. This binding site for Nod factors, termed NFBS1, which is present in both a leguminous and a non-leguminous plant, may have a more general role than symbiosis.

Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti.

In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the 35S-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulphate ([35S]PAPS) from ATP and [35S]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM. Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.