Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development.

Striga is a root parasitic weed that attacks many of the staple crops in Africa, India and Southeast Asia, inflicting tremendous losses in yield and for which there are few effective control measures. Studies of parasitic plant virulence and host resistance will be greatly facilitated by the recent emergence of genomic resources that include extensive transcriptome sequence datasets spanning all life stages of S. hermonthica. Functional characterization of Striga genes will require detailed analyses of gene expression patterns. Quantitative real-time PCR is a powerful tool for quantifying gene expression, but correct normalization of expression levels requires identification of control genes that have stable expression across tissues and life stages. Since no S. hermonthica housekeeping genes have been established for this purpose, we evaluated the suitability of six candidate housekeeping genes across key life stages of S. hermonthica from seed conditioning to flower initiation using qRT-PCR and high-throughput cDNA sequencing. Based on gene expression analysis by qRT-PCR and RNA-Seq across heterogeneous Striga life stages, we determined that using the combination of three genes, UBQ1, PP2A and TUB1 provides the best normalization for gene expression throughout the parasitic life cycle. The housekeeping genes characterized here provide robust standards that will facilitate powerful descriptions of parasite gene expression patterns.

The Use of Arabidopsis to Study Interactions between Parasitic Angiosperms and Their Plant Hosts.

Parasitic plants invade host plants in order to rob them of water, minerals and nutrients. The consequences to the infected hosts can be debilitating and some of the world's most pernicious agricultural weeds are parasitic. Parasitic genera of the Scrophulariaceae and Orobanchaceae directly invade roots of neighboring plants via underground structures called haustoria. The mechanisms by which these parasites identify and associate with host plants present unsurpassed opportunities for studying chemical signaling in plant-plant interactions. Seeds of some parasites require specific host factors for efficient germination, thereby insuring the availability of an appropriate host root prior to germination. A second set of signal molecules is required to induce haustorium development and the beginning of heterotrophy. Later stages in parasitism also require the presence of host factors, although these have not yet been well characterized. Arabidopsis is being used as a model host plant to identify genetic loci associated with stimulating parasite germination, haustorium development, and parasite support. Arabidopsis is also being employed to explore how host plants respond to parasite attack. Current methodologies and recent findings in Arabidopsis - parasitic plant interactions will be discussed.

Influence of glyphosate on amino acid composition of Egyptian broomrape.

The parasitic plant broomrape is entirely dependent on its host for reduced carbon and nitrogen and is also susceptible to inhibition by glyphosate that is translocated to the parasite through a host. Studies were conducted to examine the effect of broomrape parasitism on amino acid concentrations of two hosts: common vetch that is tolerant of low levels of glyphosate and oilseed rape that has been genetically engineered for glyphosate resistance. The influence of glyphosate on the amino acid content of broomrape and the two hosts was also examined. Amino acid concentrations in leaves and roots of parasitized common vetch plants were generally similar to those of the corresponding tissues of nonparasitized plants. Amino acid concentrations in broomrape were lower than those of the parasitized common vetch root. For common vetch, glyphosate applied at rates that selectively inhibited broomrape growth did not alter individual amino acid concentrations in the leaves, but generally increased amino acid levels at 0.18 kg ha-1. Glyphosate application also increased the amino acid concentrations, with the exception of arginine, of broomrape growing on common vetch and did not generally influence concentrations in leaves or roots of common vetch. In oilseed rape, parasitization by broomrape generally led to higher amino acid concentrations in leaves but lower concentrations in roots of parasitized plants. Broomrape had higher amino acid concentrations than roots of the parasitized oilseed rape. Glyphosate applied at 0.25 and 0.5 kg ha-1 generally increased the amino acid concentrations in oilseed rape leaves, but the 0.75 kg ha-1 application caused the amino acid concentrations to decrease compared to those of untreated plants. In oilseed rape root the general trend was an increase in the concentration of amino acids at the two highest rates of glyphosate. Individual amino acid concentrations in broomrape attachments growing on oilseed rape were generally increased following glyphosate application of 0.25 kg ha-1. These results indicate that low rates of glyphosate alter amino acid profiles in both host and broomrape and raise questions about the regulation of amino acid metabolism in the parasite.

Expression of a defense-related 3-hydroxy-3-methylglutaryl CoA reductase gene in response to parasitization by Orobanche spp.

Orobanche spp. are angiosperms that live parasitically on the roots of other plants, and are capable of significantly reducing the yield and quality of their crop hosts. We have demonstrated that parasitization by Orobanche induces expression of hmg2, a defense-related isogene of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in tobacco. Transgenic tobacco plants expressing a construct containing 2.3 kb of the tomato hmg2 gene promoter fused to the beta-glucuronidase (GUS) reporter gene were parasitized by O. aegyptiaca. Expression of the hmg2:GUS construct was detected within 1 day following penetration of the host root by the O. aegyptiaca radicle and was localized to the region immediately around the site of parasite invasion. This expression continued and intensified over the course of O. aegyptiaca development. In addition, the hmg2:GUS expression was induced by secondary parasitization, where secondary roots of O. aegyptiaca contacted the host root at a distance from the primary attachment site. This GUS expression was specific to plants containing the hmg2:GUS construct, and was not observed in control plants transformed with a construct of the cauliflower mosaic virus 35S promoter fused to the GUS gene. These results indicate that Orobanche parasitization initiates rapid and sustained induction of a defense-related gene in the host root.

Structural features of the antibody-A chain linkage that influence the activity and stability of ricin A chain immunotoxins.

The importance of the various structural elements constituting a ricin A chain immunotoxin to the stability of the disulfide bond between the antibody and A chain was examined using a panel of immunoconjugates prepared with the mouse monoclonal antibody Fib75. Analogues of the standard ricin A chain immunotoxin prepared with the N-succinimidyl 3-(2-pyridyldithio)propionate disulfide cross-linker included immunoconjugates made with N-succinimidyl 4-[(iodoacetyl)amino]benzoate the thioether cross-linker; with N-succinimidyl 3-(2-pyridyldithio)butyrate, the hindered disulfide cross-linker; with a peptide spacer between the antibody and cross-linker; or with the dodecapeptide corresponding to the C-terminus of ricin A chain. The cytotoxic activities of the immunoconjugates and their susceptibility to reduction by glutathione in vitro were compared. The thioether-linked immunotoxin could not be cleaved by glutathione in vitro and had low cytotoxic potency, consistent with the requirement of a reducible disulfide linkage for activity. The hindered disulfide-linked immunotoxin was 3-fold more stable to reduction than the immunotoxin containing a standard unhindered disulfide linkage, but the cytotoxic activities of the two constructs were indistinguishable. The introduction of a flexible peptide Ala-Ala-Pro-Ala-Ala-Ala-Pro-Ala-Pro-Ala between Fib75 and the disulfide linkage introduced by SPDP had no deleterious effect on cytotoxic activity and no effect on the susceptibility of the disulfide linkage to reduction. This finding suggests that the enforced proximity of the A chain to the antibody caused by the use of a short chemical cross-linker in a conventional immunotoxin has no influence on either of these properties in this system.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoscintigraphy of squamous carcinomas of the head and neck.

A pilot study was carried out to assess the value of a radiolabeled antibody against epidermal growth factor receptor (EGFR1) in localizing tumors in patients with squamous carcinomas of the head and neck. Positive images of large tumours (greater than 3 cm diameter) were obtained in 8 of 11 patients after intravenous administration of 111indium-labeled EGFR1. Two patients gave equivocal results, while negative scans were obtained from the patient with the smallest tumor (1 cm diameter). There were no false-positive images. The success of this study in localizing relatively large squamous carcinomas indicates that the antibody should be evaluated in patients with smaller tumors to establish the limits of detection of the technique.

The effect of chelating agents on the distribution of monoclonal antibodies in mice.

The potential for altering the biodistribution of radiolabel from gallium- and indium-labeled mouse monoclonal antibodies was investigated in mice using metal chelating agents. The chelating agents used were desferrioxamine (DFO), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-di (O-hydroxyphenylacetic acid) (EDHPA), and 2,2' dipyridyl (DIPY). The mouse monoclonal antibody LICR-LON-M8 was labeled with 111In after conjugation to DTPA, and with 67Ga after conjugation to DFO. All the chelating agents except DIPY altered the biodistribution of [67Ga]citrate and [111In]citrate but did not affect the 48-hr tissue uptake of label from [111In]DTPA-M8 or [67Ga]DFO-M8, confirming the in vivo stability of the antibody conjugates. Label fixed in the tissues was inaccessible to the chelating agents, indicating that they will not be suitable for reducing the high background liver radioactivity in patients undergoing scanning with indium-labeled antibodies.

An antibody-desferrioxamine conjugate labelled with 67Ga.

The optimum conditions for producing a 67Ga labelled antibody-desferrioxamine conjugate were determined. The mouse monoclonal antibody LICR-LON-M8 was coupled to the metal chelating compound desferrioxamine (DFO) using glutaraldehyde (GLUT). A DFO:GLUT:M8 molar ratio of 500:150:1 gave an immunoreactive antibody with low amounts (less than 5%) of high molecular weight polymer. The labelling efficiency with 67 Ga was greater than 90%, with a specific activity of 2-4 MBq/mg antibody. This 67Ga labelled antibody is suitable for evaluation as a diagnostic imaging agent.

Epithelial membrane antigen: partial purification, assay and properties.

The Epithelial Membrane Antigen (EMA) has until now only been described in immunological terms and has been shown immunohistochemically to be present on a variety of human non-squamous epithelial surfaces. It is a valuable marker in diagnostic tumour pathology and enables the detection of small deposits of malignant cells in organs such as liver and bone marrow. Its discovery in soluble form in human milk has enabled a purification of the antigen from this source. The antigenic activity in the milk is spread over a wide range of mol. wts and although purification causes a general reduction in size, the antigen remains heterogeneous. Carbohydrate forms the major component of the antigen with galactose and N-acetylglucosamine as the two major sugars. The protein content of EMA is low and shows considerable variation in amino acid composition from one sample to another. A high content of inorganic material has also been found in EMA but is not due to high sulphate or phosphate levels.

Location of metastatic breast carcinoma by a monoclonal antibody chelate labelled with indium-111.

The ability of a radiolabelled monoclonal antibody, LICR-LON-M8 (M8), to locate metastatic breast carcinomas has been investigated. The scans generated by M8, either when labelled with radioiodine, or when conjugated with diethylenetriamine-pentaacetic acid (DTPA) and labelled with radioactive indium (111In), have been compared with X-rays and 99mTc-methyl diphosphonate (MDP) bone scans. All 10 patients with skeletal metastases had positive 111In-DTPA-M8 scans and the overall correlation with X-rays and MDP scans was good but varied with the region studied. By contrast, radioiodinated M8 did not detect metastases at any site. The discrepancies between 111In-DTPA-M8 images and conventional techniques may be related to the different stages in the evolution and development of the lesion at which the various techniques detect bone metastases.

Immunohistochemical demonstration of H2 antigens in mouse tissue sections.

The immunohistological demonstration of H2 antigens in cryostat sections of a wide variety of mouse tissues is reported. The purpose in developing the method was to use H2 antigens as cellular markers in studies of mouse chimeras. Monoclonal anti-H2 antibodies were used, either with a hapten-sandwich technique using biotin or arsanilate, or as direct enzyme conjugates. The direct antibody-enzyme conjugates were simpler to use, provided an intensity of specific staining which was comparable to that obtained with the hapten-sandwich systems, and gave fewer problems of background when simultaneous double staining was attempted. The results provide a description of the distribution of H2 antigens in many of these tissues. The intensity of H2 staining varied widely from tissue to tissue, but also within tissues and between individual mice of the same litter. Quantitation by autoradiography suggests that there is a fivefold variation in available H2 antigen between tissues which are stained strongly or weakly by our technique.

The limitations of the dual radionuclide subtraction technique for the external detection of tumours by radioiodine-labelled antibodies.

A dual radionuclide subtraction technique for external detection of tumours has been evaluated to determine the viability of the method for use with radioisotope labelled antibodies. A number of external scintigraphic investigations have been carried out with 131I-labelled antibodies to carcinoembryonic antigen (CEA). The investigations were performed on patients with metastatic disease known to produce CEA. The dual radionuclide subtraction technique was used to account for the blood and tissue background. The 131I-labelled antibodies were found to localise in the metastatic lesions, but the subtraction technique using 99Tcm-labelled HSA and pertechnetate gave ambiguous results, which included the production of artefacts. The ambiguities noted in the clinical results were substantiated by experimental data, which highlight the unreliability of this technique.

The site of binding of anti-CEA antibodies to tumour CEA in vivo: an immunocytochemical and autoradiographic approach.

Radiolabelled affinity-purified antibody to carcinoembryonic antigen (CEA) was injected i.v. into immune-suppressed mice carrying xenografts of human breast carcinoma. Its distribution in the tumours was examined by a combination of immunocytochemistry and autoradiography. The antibody interacted predominantly with the CEA in the extracellular tumour space, rather than on the cell membrane or cytoplasm.

Localization of human breast-carcinoma xenografts using antibodies to carcinoembryonic antigen.

Affinity-purified antibodies to carcinoembryonic antigen (CEA) have been injected into immune-suppressed mice bearing xenografts of human breast tumours. It has been shown that the antibodies localized in the tumours but not in normal tissues. The degree of tumour localization correlates with the amount of tumour CEA, and is unaffected by levels of circulating CEA or CEA/anti-CEA immune complexes.

Chemical modifications of the protein of carcinoembryonic antigen: associated changes in immunological activity and conformation.

Chemical substitution of the exposed residues of tryptophan, tyrosine, histidine and arginine in carcinoembryonic antigen (CEA), using appropriately selective reagents, caused no significant change in the capacity of the antigen to bind to anti-CEA serum. However, treatments of CEA with 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, both in the presence of guanidine HCl, caused a large reduction in binding capacity. Measurement of the circular dichroism spectra of all of the products showed that retention of conformation of the molecular correlated well with retained antigenic activity, whereas the large losses in capacity to bind to anti-CEA sera were accompanied by a probably the result of gross conformational changes. The tyrosine residues of CEA may be classified into three categories: (i) 3 freely reacting residues, (ii) 7 or 8 moderately buried residues and (iii) 15 unreactive residues.