A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Seed Development in Phaseolus vulgaris L. cv Seminole: I. Developmental Independence of Seed Maturation

Phaseolus vulgaris L. cv Seminole pods removed from the plant continued their development when incubated in suitable conditions. Seeds continued to grow and develop and pods and seeds passed through an apparently normal developmental sequence to dryness. Seed growth was at the expense of pod dry weight (DW) reserves. Losses of pod DW paralleled DW gains by seeds in detached pods and in pod cylinders containing a seed. The transfer activity was apparent only within the period 10 to 30 days after anthesis (DAA) with maximal activity between 15 to 20 DAA. This period corresponds to maximum pod growth and the attainment of maximal DW. Seeds are in only the early phase of seed growth at this time. No DW transfer was observed at developmental stages beyond 30 to 35 DAA when normal senescence DW losses in pods became evident and seeds were in the later phase of seed fill. Pods or pod cylinders remained green and succulent over the transfer period, later passing through yellowing and drying phases characteristic of normal development. DW transfer was dependent on funicle integrity and was readily detectable in pod cylinders after 7 days incubation. The DW transfer activity may contribute to continuing nutrition of seeds under conditions where the normal assimilate supply to seeds becomes limiting. Defoliation and water stress treatments applied to Phaseolus plants reduced seed yields but allowed persistence of seed maturation processes such that all seeds developing to dryness were capable of germination.     

Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

The localization of neutralization determinants within the envelope glycoproteins of human immunodeficiency virus (HIV) has been largely achieved by immunizing small animals in conjunction with Freund's adjuvant. However, for eventual use in humans, candidate HIV vaccine components must also be efficacious in a nontoxic formulation. We describe here the production of hybrid Ty viruslike particles carrying the major neutralizing domain of HIV and demonstrate the induction of high-titer virus-neutralizing antibodies and an HIV-specific T-cell proliferative response after immunization in conjunction with aluminum hydroxide. As aluminum hydroxide and aluminum phosphate are the only adjuvants currently licensed for use in humans, these observations have implications for the development of an effective vaccine against HIV.     

THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PYRENOID PROTEIN OF EREMOSPHAERA VIRIDIS

The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient centrifugation and their physical and enzymatic properties were studied. The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid. Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels. The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2:1. The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein. Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components. The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein. The pyrenoid was found to contain a high specific activity of ribulose-1,5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations. It is suggested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway.     

Toward Patient-Specific,Biologically Optimized Radiation Therapy Plans for the Treatment of Glioblastoma

Purpose

To demonstrate a method of generating patient-specific, biologically-guided radiotherapy dose plans and compare them to the standard-of-care protocol.

Methods and Materials

We integrated a patient-specific biomathematical model of glioma proliferation, invasion and radiotherapy with a multiobjective evolutionary algorithm for intensity-modulated radiation therapy optimization to construct individualized, biologically-guided plans for 11 glioblastoma patients. Patient-individualized, spherically-symmetric simulations of the standard-of-care and optimized plans were compared in terms of several biological metrics.

Results

The integrated model generated spatially non-uniform doses that, when compared to the standard-of-care protocol, resulted in a 67% to 93% decrease in equivalent uniform dose to normal tissue, while the therapeutic ratio, the ratio of tumor equivalent uniform dose to that of normal tissue, increased between 50% to 265%. Applying a novel metric of treatment response (Days Gained) to the patient-individualized simulation results predicted that the optimized plans would have a significant impact on delaying tumor progression, with increases from 21% to 105% for 9 of 11 patients.

Conclusions

Patient-individualized simulations using the combination of a biomathematical model with an optimization algorithm for radiation therapy generated biologically-guided doses that decreased normal tissue EUD and increased therapeutic ratio with the potential to improve survival outcomes for treatment of glioblastoma.     

Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.     

Crescentic glomerulonephritis--a manifestation of a nephritogenic Th1 response?

Crescentic glomerulonephritis (GN) is the histopathological correlate of the clinical syndrome of rapidly progressive glomerulonephritis. Glomerular crescent formation complicates proliferative forms of GN and indicates severe disease with a poor renal prognosis. In the past 10 years evidence from experimental models of GN and from human disease has accumulated suggesting that crescentic glomerulonephritis is a manifestation of a delayed type hypersensitivity (DTH)-like response to nephritogenic antigens. The elucidation of T helper 1 (Th1) and Th2 subsets in mice and in humans has led to the hypothesis that crescentic GN is a manifestation of a Th1 predominant DTH mediated immune response. Recent experiments performed mainly in a murine model of crescentic glomerulonephritis have tested this hypothesis. Crescent formation in this model is substantially interleukin (IL)-12 and interferon-gamma (IFN-gamma) dependent. Administration of IL-12, deletion of endogenous IL-4 or IL-10 results in enhanced disease, while administration of exogenous IL-4 and/or IL-10 reduces crescentic injury. These findings, together with the available evidence from human studies (examining the pattern of immune effectors in glomeruli, data on cytokine production by peripheral blood mononuclear cells and case reports of the induction of proliferative and/or crescentic GN by administration of IFN-gamma or IL-2) suggest that human crescentic GN is manifestation of a Th1 mediated DTH-like nephritogenic immune response.