A constitutive gene expression system derived from the tCUP cryptic promoter elements.

A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops ( Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5' untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a beta-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed

Implementing real-time system interfaces to support the shared care of diabetes patients.

This paper describes a collaborative project: the Medway Integrated Care Support (MEDICS) Project, involving GP and Hospital system suppliers, an NHS Trust, General Practitioners and the NHS Information Authority. The objective of the project is to assess the Object Management Group's (OMG/CORBAMed) Clinical Observations Access Service (COAS) standard, by demonstrating its use in implementing a real-time interface between a GP system and a hospital clinical system, supporting the shared care of diabetes patient.

Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean.

Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and ß-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected in the T7 generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27-92%, but transformation events were usually restricted to non-regenerable callus.

Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes.

Eighteen of 20 short-season soybean (Glycine max (L.) Merrill) genotypes (maturity group 0 and 00) screened for proliferative embryogenic capacity formed secondary globular embryos, at rates of 1-70% of cultured immature cotyledons. Five genotypes produced embryogenic cultures which were proliferative for at least 6 months. Proliferative embryogenic cultures of AC Colibri and X2650-7-2-3 were bombarded using a Bio-Rad PDS-1000/He particle gun. Co-bombardments with plasmid pairs pHygr (encoding a type IV aminoglycoside phosphotransferase;aphIV) and pRD300pat (encoding a phosphinothricin N-acetyltransferase;pat) or pRD300pat and pFF19G (ß-glucuronidase;uidA or gus) resulted, respectively, in 12 hygromycin-selected lines with multiple insertions of aphIV and pat, and two l-phosphinothricin-selected lines plus three ß-glucuronidase-positive lines recovered without selection. Although fertile plants were recovered from young proliferative cultures, transgenic plants, which were derived from cultures 12-14 months of age, were sterile.

High frequency production of doubled haploid plants of Brassica napus cv. Topas derived from colchicine-induced microspore embryogenesis without heat shock.

This report describes a very high genome doubling efficiency of Brassica napus cv. Topas plants, derived from microspores induced to undergo embryogenesis with a colchicine treatment, without the use of a heat treatment. The plants showed normal growth and development, and 90% were fertile. In contrast, only 6% of the plants derived from heat-induced embryos were fertile diploids. All cytological analysis of the progeny of fertile plants showed 2n=38 chromosomes. These results show that colchicine can simultaneously induce microspore embryogenesis and double the ploidy level to produce doubled haploid plants.

The use of granulocyte colony-stimulating factor to deliver four cycles of ifosfamide and epirubicin every 14 days in women with advanced or metastatic breast cancer.

Twenty patients with locally advanced or metastatic breast cancer were treated with four cycles of ifosfamide/mesna 5 g m-2 and epirubicin 60 mg m-2 every 14 days with granulocyte colony-stimulating factor (G-CSF, Filgrastim). Complete remission occurred in six out of the 20 patients (30%, 95% confidence interval 12-54%) and there were 12 partial responders (60%, 95% confidence interval 37-81%), thus giving an overall response rate of 90% (95% confidence interval 63-97%). Two patients had progressive disease. The median duration of response for those patients with metastatic disease was 7.3 (1.3-20.1+) months. The median survival time for these patients was 15 (5.3-27.9+) months. Of the four patients treated with locally advanced disease three achieved a complete clinical response and one a partial response. Three out of four of these patients subsequently underwent a mastectomy, and in one of these no viable tumour was seen. Our conclusion is that this regimen is excellent palliation for metastatic disease and possibly useful neoadjuvant treatment.

Pharmacokinetics, metabolism and clinical effect of ifosfamide in breast cancer patients.

Ifosfamide (IFO) at a dose of 5 g/m2, was administered as a 24-h infusion to 15 patients with metastatic (12) or locally advanced (3) breast cancer (age range 33-59 years, median 46). Concurrent chemotherapy was doxorubicin (40 mg/m2) or epirubicin (60 mg/m2). Ifosfamide and its metabolites were measured in plasma and urine during and for 24 h after the infusion using a high performance thin layer chromatography (HPTLC) technique. Patients' haematological toxicity and biochemistry were monitored during treatment and patients were followed for up to 2 years after therapy. At the time of evaluation, 5 of the patients were alive, 2 of whom had not relapsed. A marked variation was observed in the pharmacokinetics and metabolism of ifosfamide in the evaluable patients. Clearance, volume of distribution and half-life of the drug were 3.48 +/- 0.88 1/h/m2, 0.56 +/- 0.22 l/kg and 4.68 +/- 2.01 h, respectively. There was no apparent correlation between these pharmacokinetic variables and patient age, weight or renal function. AUCs of the ultimate alkylating species isophosphoramide mustard (IPM) varied over 6-fold, as did those of the inactivated metabolite carboxyifosfamide (CX). AUCs of dechloroethylated metabolites varied 4-fold (3-dechloroethylifosfamide, 3-DCI) or 8-fold (2-DCI), while that of the parent compound varied only 2.5-fold. Variation in recovery of the metabolites in urine varied over an even wider range, total recovery varying from 17.5 to 81.8% of the dose administered. There was little apparent correlation between pharmacokinetic and metabolite parameters of IFO and haematological toxicity. However, there was a marked negative correlation between both progression-free interval and survival and the AUCs of the products of IFO activation (IPM and CX). In addition, the recovery of IPM in urine was higher in patients experiencing a partial response compared to those with progressive or stable disease. Recovery of dechloroethylated metabolites correlated positively with survival, if 1 poor prognosis patient was excluded. Although far from conclusive, these results give some insight into a possible mechanism of action of ifosfamide and indicate that some species other than IPM, as measured systemically, is responsible for the pharmacological effects of this drug.

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.

Standards for medical graphics.

A lack of understanding of simple standards is responsible for poor quality graphics in slides, overhead projection transparencies, posters and publications. The ready availability of computer graphics application programs has led to a proliferation of well-executed, but poorly constructed illustrations. Guidelines are given for the design of scientific illustrations, with reference to correct labelling, line weights, font styles and sizes, the use of colour, and other factors affecting the clarity of presentation. The application of established standards for the preparation of medical graphics as described is strongly recommended.

Effects of culture density, conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas.

In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2-4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1-4 days or 0-4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.

Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh.

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20-40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.

Prophase bands of microtubules occur in protoplast cultures of Vicia hajastana Grossh.

Circumnuclear bands of microtubules (MT) have been found in the prophase of mitoses in cultured protoplasts of Vicia hajastana. The timing of the appearance and disappearance of the prophase band of MT (PB) relative to the stage of mitosis was studied using simultaneous staining of MT by immunofluorescence and DNA by Hoechst 33258. These protoplasts regenerate into unorganized tissue. Pre-prophase bands of MT have previously been found only in highly organized tissues of higher plants. The role of PB in cell division is discussed.

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells.

Various modifications to the immunofluorescent labeling procedures for microtubules in plant cells have been compared using cell cultures of Vicia hajastana Grossh. Using serial section electron microscopic reconstructions as a reference, we have chosen as our standard procedure a method that maximizes both the preservation of the cytoskeleton and the proportion of cells staining, while minimizing the degree of nonspecific staining. The critical steps of the procedure include stabilization of the cytoskeleton, cell wall permeabilization, and cell extraction. To maintain structural integrity during the procedure, it is necessary to stabilize the cytoskeleton with paraformaldehyde. To facilitate antibody penetration into the cell, it was necessary that the walls be made permeable via partial enzymatic digestion. Detergent extraction of cells increased the proportion of cells staining and decreased the level of nonspecific binding of the antibodies. The procedures detailed in this article provide a good starting point for the application of immunofluorescent labeling techniques to other plant systems.

How to: Produce a Good Poster.

'Poster sessions' have become a widespread feature of medical meetings. Here, an experienced medical artist gives his guidelines for producing a poster that provides clear information in an attractive and easy-to-assemble format.

Organization of microtubules in dividing and elongating cells of Vicia hajastana Grossh. in suspension culture.

Cells of Vicia hajastana Grossh. cultured with 2,4-D showed coupled division and growth and formed multicellular files of small isodiametric cells. In GA without added 2,4-D, the cells stopped dividing and continued elongating for several days. Total growth was the same in both hormone conditions. An immunofluorescent technique was developed to study microtubule (MT) distribution. Cells in GA showed parallel MT arrays oriented transversely to the axis of elongation. In some cells the number of MT per unit length was maintained during growth while other elongating cells showed reduced frequency of MT. Microtubules often appeared as thickened, branched strands, probably as a result of lateral aggregation. In cells grown in 2,4-D some pre-prophase bands of MT were observed. Cells in mitosis lacked cortical MT, and all organized staining was in spindles or phragmoplasts. Interphase cells in 2,4-D showed variable organization of cortical MT ranging from disordered to transversely ordered. Cells in early interphase had disordered MT while larger cells showed order. These observations indicate that MT in cycling cells are continually changing organization, probably accounting for the different distributions observed in interphase cells. On cessation of the mitotic cycle, reorganization of MT stops and transverse arrays of cortical MT are maintained as cells elongate. These processes are similar to those observed in organized tissues; however, cultured cells offer distinct advantages for experimental manipulation and microscopic observation of cytoskeleton.

NMR anatomy of the brain using inversion-recovery sequences.

The use of NMR inversion-recovery (IR) sequences to demonstrate brain anatomy is illustrated. The high level of grey-white matter contrast is of value in localising anatomical structures and demonstrating myelination during childhood. While the resemblance of IR scans to gross anatomical sections in different planes is close, it is limited by the spatial resolution of the NMR scanner, artefacts and partial volume effects.