Biomedical subjects
Publications and source records attributed to J Cullimore.
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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.
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.
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.
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.
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.
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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.
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A series of seven women aged 50 years and over was studied over two years to analyse patient profile and pregnancy outcome. All were first-generation immigrant Asian multigravidae who were Muslims of low socio-economic status and who had never used contraception. The median age at booking was 52 (range 51-59 years) and the median parity was nine. One woman had gestational diabetes and one had an antepartum haemorrhage. There were few intrapartum or puerperal problems but an increased rate of Caesarean section (28.6%) was noted. There were no stillbirths, neonatal deaths or congenital anomalies in this series. The mean birth weight was 3.3 kg and the mean gestation at delivery was 39.2 weeks. There was no significant perinatal morbidity. Five of the seven women declined further family planning advice. A possible relationship between race, parity and age at menopause is discussed.
OBJECTIVE: To report the use of the Cardial inferior vena caval filter as prophylaxis against pulmonary embolism in pregnant women with extensive iliofemoral thrombosis. SETTING: Leicester Royal Infirmary. SUBJECTS: Four pregnant women with extensive iliofemoral thrombosis, deemed to be at high risk of pulmonary embolism, managed over a period of one year. TECHNIQUE: In addition to standard full anticoagulation with heparin, the Cardial inferior vena cava filter was introduced percutaneously under local anaesthesia through the unaffected contralateral femoral vein and positioned in the inferior cava below the renal veins. RESULTS: The procedure was uncomplicated and did not compromise feto-maternal condition. There was no evidence of pulmonary embolism after filter insertion. CONCLUSION: The use of inferior vena cava filters should be considered as an adjunct to intravenous anticoagulation in pregnant women with extensive deep vein thrombosis of the lower limbs.
Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.
OBJECTIVE: To determine the efficacy and morbidity of fine loop diathermy excision of the cervical transformation zone as applied to the management of outpatients with abnormal cervical smears. DESIGN: Prospective programme trial with six month follow up. SETTING: Two hospital based colposcopy clinics. PATIENTS: 616 Patients aged 16-60 with abnormal cervical smears. INTERVENTIONS: After colposcopic and cytological assessment excision of the cervical transformation zone by fine loop diathermy under local anaesthesia in the outpatient department. MAIN OUTCOME MEASURES: Time to complete the treatment, immediate morbidity in terms of discomfort and bleeding, and cytological and colposcopic findings at six months. RESULTS: Treatment was completed in a mean of 3.47 minutes (SD 1.99). Immediate morbidity was minimal, and histological specimens were adequate in over 90% of cases. Almost two thirds of patients were treated at their first visit to the clinic. 58 Patients (9.4%) failed to attend for follow up at six months and one had had a hysterectomy. Of the 557 patients who attended for colposcopic and cytological follow up at six months, 506 (91%) were normal cytologically and 19 (3.4%) had histologically confirmed persistence of cervical intraepithelial neoplasia. The overall confirmed failure rate of the technique was 4.4%. CONCLUSION: Loop diathermy excision is an effective treatment with low morbidity and is an appropriate modality for patients with abnormal cervical smears.
The performance of a new ectocervical brush sampler--the Cervex--was compared with the Ayre spatula in 280 paired cervical smears. The Cervex smears were superior in quality of spread, transformation zone sampling in all degrees of cervical patency and in detection of histologically proven epithelial abnormalities, with a false negative rate of 10.9% compared with 20% for the Ayre. Improvement in predictive value was noted in atrophic samples, with increased cellularity and transformation zone representation. Difficulty has been encountered in obtaining adequate samples from the older woman and from those with iatrogenic scarring of the cervix. Although two-sampler techniques may be used, submission of high quality pan-cervical material from a single sampler onto one slide is economically and organizationally attractive. The Cervex seems capable of producing such samples and deserves further evaluation for routine screening.
Cervical squamous carcinoma in situ with early stromal invasion has recently been defined as a strict histologic entity. Two cases of extensive classical adenocarcinoma in situ of the uterine cervix and one of very early invasive adenocarcinoma are reported that showed foci indistinguishable from squamous early stromal invasion arising directly from unequivocally glandular in situ malignant foci. This finding is further evidence in support of the theory that glandular and squamous intraepithelial neoplasia in the cervix are closely related conditions, possibly with a common origin in the subcolumnar reserve cell.
The paper describes the technique of 'LLETZ' (large loop excision of the transformation zone), a new method of management for women with an abnormal cervical smear which offers the advantages of conization with those of local destruction. A large loop of thin wire forms a diathermy electrode that allows deep excision of the transformation zone with minimal tissue damage. The tissue removed can be examined histologically. The technique was used to investigate and treat 111 women with abnormal smears referred to the Bristol Royal Infirmary during 1986. Microinvasive disease was revealed in one woman where it was not suspected by cytology or colposcopic examination. Of 102 women followed up for at least 1 year by cytology, colposcopy and, where appropriate, histology, two women were found to have residual/recurrent cervical intraepithelial neoplasia.
Treatment of cervical intraepithelial neoplasia, both squamous and glandular, is based on the premise that this condition may ultimately progress to invasive cancer. Treatments have evolved in a conservative direction resulting in reduced morbidity yet also achieving satisfactory control of disease. As yet there is no way in which those cases that have malignant potential can be distinguished from those that do not. Satisfactory management is dependent upon the exclusion of an invasive process. Cytologic, colposcopic and histologic confirmation of preinvasion are therefore natural precedents to management.