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Contains fulltext : 50113.pdf (Publisher’s version ) (Closed access) A multiplexed fluorescence immunoassay using a novel planar waveguide technology-based microarray system, ZeptoMARK (Zeptosens), was developed to detect simultaneously urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues. The three analytes assay was cross-validated with single-analyte ELISA/chemiluminescence immunosorbent assay tests, revealing good correlations and enhanced assay sensitivities (LODs) of 1 pg/mL for uPA, 33 pg/mL for PAI-1, and 1 pg/mL for VEGF. Values were well within the 80-120% limits for assay recovery and within the +/-20% limits for assay precision. The uPA, PAI-1, and VEGF results obtained from 50 breast cancer cytosols using the protein array system demonstrated that the microarray-based multiplexed assay is a sensitive and robust tool to be used for the simultaneous quantification of cancer markers in small breast cancer tissue samples (core biopsies). The miniaturized, multiplexed assay format has a potential to be used for the quantitative analysis of a larger set of validated markers with significance in disease management.

CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c -type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome c maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.

Given the influence of photoperiod on reproductive development and whole-plant senescence in monocarpic plants, one would suspect that leaf senescence in these plants might be under photoperiodic control. In Arabidopsis thaliana, which is monocarpic and also a nonobligate long-day (LD) plant, LDs (16 h, 300 μmol m−2 s−1) caused leaves to die earlier than did short days (SDs, 10 h). Since leaf longevity was not paralleled by the reproductive development in the present study, the reproductive structures did not seem to be the primary controls of leaf senescence. The LD effect appeared to depend on the amount of light rather than on day length, for leaves given LDs at reduced light intensity (180 μmol m−2 s−1) lived longer than those in LDs with full light. In addition, the higher light intensity promoted chlorophyll loss and anthocyanin accumulation in LDs. Thus, senescence of these leaves seems to be governed by light dosage rather than photoperiod. Light may play a natural role in promoting the senescence of A. thaliana leaves.

Publisher Summary This chapter focuses on the light control of leaf senescence. Light control of leaf senescence reflects important environmental controls of plant development and has many practical applications as well. Light is required for the photosynthesis that sustains plant growth and long-term survival and also has many practical applications. The postharvest longevity of green vegetables and leaf-bearing cut flowers are influenced by the light signals. Lack of light for photosynthesis causes starvation and thereby senescence, and death. Although the induction of senescence by darkness is an extreme and unnatural case, photosynthesis and light excitation pressure are involved in the senescence-delaying effects of light. Light dosage effects on leaf senescence are best viewed relative to the CO 2 compensation point that light intensity where photosynthetic CO 2 fixation equals or compensates for the release of CO 2 by respiration.

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000, 97, 9390-9395) [1], most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bio-analytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the ‘wild type’ (HL50). The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.

Seeds of Nigella damascena L. germinated in darkness. Incandescent light inhibited germination, with greater inhibition occurring during the latter half of the 24-h imbibitional period. The inhibitory effects of incandescent radiation depended upon irradiance; inhibition of germination by a 12-h irradiation also exhibited a broad spectrum FR irradiance dependence. An action spectrum in the region of 650-740 nm revealed that 720-nm radiation was most inhibitory, suggesting that a HIR was operative. An action of P, evidenced by photoreversible control of germination following brief R and FR irradiations, was documented when temperature was shifted upward but not when temperature was held constant.

Standardized culture methods for bryophyte plants, tissues, and propagules were developed. The methods overcome many past inconveniences and offer certain advantages that justify a broader use of bryophyte material for fundamental physiological and biochemical research. Five substrates prove to be especially advantageous for the culture of the liverwort, Marchantia polymorpha. They include vermiculite, perlite, glass cloth, nutrient agar, and nutrient solution. Selected culture conditions and vessels especially adapted to each substrate are described. The methods are contrasted for ease of culture maintenance, yields, and amenability to specific experimentation.

A naturally occurring growth regulator in extracts of Marchantia polymorpha tissues has been characterized by paper chromatography and bioassay. The endogenous compound is presumed to be indoleacetic acid (IAA) on the basis of its chromatographic behavior in five solvents, its biological activity in the Avena straight-growth test, and the utilization of synthetic IAA by thallus tissues. The endogenous level of IAA in M. polymorpha thalli was estimated. The results from the sufficiently rigorous methods employed establish for the first time the presence of IAA in a bryophyte species.

Edstrom's microphoresis technique has been employed to determine the quantitative alterations in nucleic acid content and base composition of individual cells associated with the initiation of bud primordia in Funaria hygrometrica. The filamentous protonema of this moss initiates bud cells which through repeated divisions form the leafy gametophores. The cytokinin, 6-furfurylaminopurine (kinetin), was used to induce the differentiation of bud cells from protonematal cells. The total RNA content of kinetin-induced bud cells (22.0 mumug/cell) was nearly 15 times that of protonematal cells (1.6 mumug/cell). The same dramatic increase in total RNA was apparent in bud cells which developed spontaneously in older cultures. As would be predicted, the adenine (A) to guanine (G) ratio for DNA from bud and protonematal cells was identical (0.7). The A:G ratio for RNA from bud cells (1.0) was much lower than that from protonematal cells (1.7). Thus, kinetin-induced differentiation in this system involves a dramatic increase in total RNA, the base composition of which approaches that of DNA. The base composition (A:G ratio) of DNA remains constant.

IN mammalian cells, hyperthermia causes temperature-dependent changes in cell growth parameters1, reduces DNA and protein synthesis rates2 and cell metabolism3, and leads to a loss of proliferative capacity4,5. Harris6 has shown that temperature-resistant pig kidney cells can be obtained after multiple exposures to very high thermal doses (colony-forming ability reduced to 10−5–10−6 of controls). The purpose of our experiments was to determine whether a single hyperthermic treatment (44 °C for 1 h) could induce a state of thermotolerance in cells, and if so, what was the mode of origin of the thermal resistant cells.

—Applying criteria used for higher plants, phytochrome mediation of uredospore germination in the stem-rust fungus, Puccinia graminis f. sp. tritici (Eriks & Henn) Guyot is established. A 1 min red irradiation at 660 nm promotes uredospore germination and this potentiation of promotion is photoreversible by a 1 min far-red irradiation at 730 nm.