Biomedical subjects
Publications and source records attributed to J Stiller.
We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.
Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.
Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.
Health spending is expected to resume its rise as a share of gross domestic product (GDP) in the projection period, following 6 years of near stability, increasing from 13.5 percent in 1997 to an estimated 16.2 percent by 2008. This implies an approximate doubling of health spending, from $1.1 trillion in 1997 to $2.2 trillion by 2008. We anticipate a reversal in recent patterns of growth in public and private health spending, with private spending expected to accelerate while Medicare spending slows in response to the implementation of the Balanced Budget Act (BBA) of 1997.
Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.
To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of beta-glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T2 generation and germinal excision events were scored in segregating T3 families as complete beta-glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T3 offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.
Twenty-seven consecutive patients with acute intractable headaches were treated in an office setting with an intravenous protocol of an antiemetic, hydrocortisone sodium succinate, and dihydroergotamine (DHE). All patients had significant pain relief within 45 minutes. Patients who were not pain-free post-treatment uniformly reported decreasing pain at the time of departure from the office. No patients required further treatment (including narcotic medication) for acute headache. All patients were followed for at least 24 hours. Adverse reactions were transient and mild. Akathisia was not uncommon and has probably been underestimated in previous studies.
A patient developed a painful progressive paraparesis following trauma. Metrizamide myelography revealed an arachnoid cyst. A delayed metrizamide computed tomography scan of the area confirmed the cyst and demonstrated a syrinx at that level that was not appreciated on the myelogram. The association of these two lesions after trauma are perhaps more common than appreciated and the importance of a computed tomography scan of the abnormal area on myelography is emphasized.
An enzyme immunoassay that utilizes antigen bound to a matrix which can be removed from the substrate to stop development is described. The assay which is performed in glass or plastic disposable tubes uses Gel-Bond film strips for attachment of antigen. The only equipment requirements are a rotary shaker and a spectrophotometer (optional). The antigen coated strips are passed through a series of tubes containing test serum, wash solution, antibody-enzyme conjugate, wash solution and substrate-chromogen taking about 45 minutes to perform. In testing sera with or without antibody to Brucella abortus a very high correlation existed between same day tests and tests performed over several days as well as with data on the same sera obtained by an enzyme immunoassay in a microtiter format.
An enzyme immunoassay for determination of individual isotype concentrations in bovine serum was developed. Polystyrene tubes were coated with affinity purified goat antibovine IgA, IgG1, IgG2 or IgM, washed and then incubated with purified isotypes to ascertain crossreactivity and sensitivity limits. Bound isotype was detected using the homologous affinity purified antibody conjugated to horseradish peroxidase and hydrogen peroxide-2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate/chromogen. A standardized serum was diluted and used as a control for comparison. Several dilutions were used initially, however, determinations may be made with a single dilution, 1:200, for all isotypes. Results for 100 sera were compared to data obtained with the same samples using a radial immunodiffusion technique. A low correlation coefficient was noted between results from the two assays. Day to day variation and within test repeatability were determined for both assays using ten samples. For the enzyme immunoassay method, day to day variation for IgA, IgM, IgG1 and IgG2 determinations was 17.5, 19.3, 7.6 and 7.3% while variation in repeatability (within a test) was 6.2, 5.9, 3.3 and 4.5%, respectively. Day to day variation for the single radial immunodiffusion test for IgA, IgM, IgG1 and IgG2 was 15.4, 26.0, 11.5 and 18.3% and variation repeatability (within a test) was 11.6, 13.9, 5.9 and 8.3%, respectively. The procedures consistently detected 0.1 micrograms of immunoglobulin whereas the radial diffusion sensitivity limit was approximately 500 micrograms.
Bovine complement was treated with various agents known to activate or inactivate one or more of the cascade components. The treated complement was then assessed for remaining hemolytic activity by a tube titration test and a radial hemolysis method. Divalent cation chelators (EDTA and EGTA); immune complexes prepared with serum and IgM, IgG1, IgG2, and IgA isotypes; smooth and rough lipopolysaccharides and lipid A; hydrazine; zymosan; cobra venom factor and brown recluse spider venom caused depletion of complement as determined in the tube titration test. Immune complexes (prepared with serum); hydrazine; cobra venom factor; EDTA and smooth lipopolysaccharide caused loss of hemolytic activity in the radial hemolysis test. This evidence suggests that the radial hemolysis test assessed complement consumed by the alternate pathway, while the tube titration method measured classical pathway consumption.
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Two IgG1 fragments were isolated from bovine colostral whey. Based on gel diffusion analysis, both fragments originated from the Fc portion of the molecule but were not identical to IgG1 Fc prepared by papain cleavage. The molecular weights were determined to be 66,000 and 14,000 daltons and it was hypothesized that the larger fragment could be a polymer of the smaller. No IgG1 Fab or IgG2 fragments could be demonstrated.
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Various buffers (0.2 M acetate, pH 4.2; 0.01 M phosphate with 0.15 M sodium chloride, pH 7.2; 0.15 M borate, pH 8.2 and 0.025 M carbonate, pH 9.6) were used as coating buffers with Brucella abortus alkali treated lipopolysaccharide on two types of plastic matrices. Maximum binding occurred using the phosphate buffer. However, as was the case with the other buffers 50 per cent or more of the antigen was removed by five washing procedures. B abortus S19 or S2308, suspended in ammonium acetate-carbonate buffer, pH 8.2, was shown to bind maximally when 10(9) cells were dried onto the plastics. Less than 20 per cent of the cells were removed by five washings.
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