Antibodies against individual thylakoid membrane proteins as molecular probes to study chemical and mechanical freezing damage in vitro

The release of proteins and the loss of biochemical activities under mechanical and chemical stresses during freezing of isolated thylakoid membranes were investigated, using polyacrylamide gel electrophoresis, single radial immunodiffusion and the measurement of cyclic photophosphorylation. Antibodies against purified proteins derived from the stromal (coupling factor CF1, ferredoxin-NADP⁺ reductase) and the lumenal side (plastocyanin) of the membrane vesicles were used as probes. Low initial solute concentrations were employed to generate mechanical stress. Chemical stresses were manipulated by varying the molar ratios of cryotoxic to cryoprotective solutes at high initial solute concentrations. Constant low amounts of ferredoxin-NADP⁺ reductase were lost from the membranes during freezing, irrespective of the composition of the suspending media. Damage at high initial osmolalities was accompanied by the release of CF1, which was influenced by the ratio of potentially cryotoxic to cryoprotective solutes, as demanded by the colligative theory of membrane cryopreservation. CF1 release and loss of cyclic photophosphorylation were linearly correlated at different ratios of salt to sucrose. However, the correlation data revealed that CF1 release could account for only part of the observed cryoinjury. Plastocyanin release was predominant at low initial osmolalities and was not influenced by the chemical composition of the suspending media. This indicates mechanical damage by membrane rupture. Under these circumstances loss of plastocyanin and loss of cyclic photophosphorylation were linearly correlated. Loss of photophosphorylation could be prevented by the addition of up to 1.2 mg plastocyanin/ml prior to freezing. It could also be ameliorated to a large extent by raising the phenazine methosulfate concentration in the test assay from 30 to 230 μM. This indicates that the membranes are able to reseal after rupture, maintaining a proton gradient upon illumination and that it is the loss of plastocyanin from their lumen that inhibits cyclic photophosphorylation.     

Antibodies to synthetic peptides as probes of acetylcholine receptor structure

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha-chain. Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa). Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9-10 kDa). No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.     

Use of PROTACS as molecular probes of angiogenesis

Small molecules designed to specifically activate or inactivate protein functions have been useful to study biological processes. PROTACS are small molecule chimera which comprise a ligand and a peptide recognition motif for an E3 ligase. These novel reagents exploit the ubiquitin-mediated proteasome degradation pathway to target the ligand-bound protein for intracellular degradation. Here, we report that an estrogen receptor (ER)-targeting PROTACS that causes degradation of ER is able to potently inhibit endothelial cell differentiation in a three-dimensional angiogenic sprouting assay. These findings support the use of ER-targeting PROTACS as probes of angiogenesis.     

Antibodies as probes of cellular differentiation and cytoskeletal organization in the mouse blastocyst

Indirect immunofluorescence microscopy has been used to detect cytoskeletal proteins, which allow a distinction between the two cell types present in the mouse blastocyst: i.e. the cells of the inner cell mass (ICM) and the outer trophoblastic cells. Antibodies against three classes of intermediate-sized filaments (cytokeratins, desmin and vimentin), as well as antibodies against actin and tubulin were studied. Antibodies against prekeratin stain the outer trophoblastic cells but not the ICM in agreement with the findings on adult tissues that cytokeratins are a marker for various epithelial cells. Interestingly, vimentin filaments typical of mesenchymal cells as well as of cells growing in culture seem to be absent in both cell types of the blastocyst. Thus, the cytokeratins of the trophoblastic cells seem to be the first intermediate-sized filaments expressed in embryogenesis. Antibodies to tubulin and actin show that microtubules and microfilaments are ubiquitous structures, although microfilaments have a noticeably different organization in the two cell types. In addition, since early embryogenic multipotential cells show close similarities to teratocarcinomic cells, a comparison is made between the cells of the blastocyst, embryonal carcinoma cells (EC cells) and an epithelial endodermal cell line (PYS2 cells) derived from EC cells. EC cells display vimentin filaments whereas PYS2 cells show both vimentin and cytokeratin filaments. The results emphasize the usefulness of antibodies specific for different classes of intermediate filaments in further embryological studies, and suggest that cells of the blastocyst and EC cells differ with respect to vimentin filaments.     

Biotinylated oligonucleotides as probes in the method of molecular hybridization

The chemical approaches to the preparation of non-radioactive and biotinylated probes based on synthetic oligonucleotides and their applicability to the molecular hybridization method are discussed.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

DNA aptamers as molecular probes for colorectal cancer study

Background

Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s) underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development.

Methodology and Results

Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh), nor do they recognize most other cancer cell lines tested.

Conclusion/Significance

The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.     

Synthesis of unimolecularly circular G-quadruplexes as prospective molecular probes

Synthesis of unimolecularly circular G-quadruplex has been accomplished for the first time during our investigation on the template basis of G-quadruplex through chemical ligations of guanine-rich linear sequences of oligodeoxyribonucleotides. The uniqueness of this newly designed circularization course is its self-recognition and self-templating on the scale of individual strand of oligodeoxyribonucleotide in which the same linear sequence serves both as a template and as a substrate simultaneously. The results from our exonuclease and DNAse hydrolysis studies confirm that there is indeed absence of open termini within the structure of the identified circular product. Our subsequent investigation on the loop-size effect indicates that the unimolecularly circular G-quadruplex possessing two or more thymine nucleotides within their connecting loops is readily attainable, while the linear sequence with a single thymine nucleotide between guanine tracts is not a proper precursor for our ligation reaction. In addition, conformation dependency of the circularization course as well as the effects of alkali ions, pH values and concentration of potassium ions on the circularization reaction are examined during our investigation. The implication of our current studies and possible application of the obtained unimolecularly circular G-quadruplex in certain biological processes are also discussed in this report.     

Antibody-nucleic acid interactions. Antibodies to psoralen-modified RNA as probes of RNA structure.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.     

Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes

The compound eye of D. melanogaster is a reiterative pattern of facets, each containing eight photoreceptor cells in a precise arrangement. This pattern is established in the eye imaginal disc during the third larval instar. A wave of morphogenesis sweeps from posterior to anterior across the disc, leaving in its wake organized clusters of photoreceptor cells. We have used monoclonal antibodies to highlight pattern elements that are not readily observable by other techniques. Monoclonal antibodies can be used to identify the molecules associated with particular patterns, providing links between observable structures and the genes. As an example, we present the purification and N-terminal sequence of a glycoprotein antigen specific to photoreceptor cells and their axons.     

The molecular genetics and neurobiology of developmental dyslexia as model of a complex phenotype

Among complex disorders, those concerning neuropsychiatric phenotypes involve particular challenges compared to disorders with more easily distinguished clinical signs and measures. One such common and unusually challenging phenotype to disentangle genetically is developmental dyslexia (DD), or reading disability, defined as the inability to learn to read and write for an otherwise normally intelligent child with normal senses and educational opportunity. There is presently ample evidence for the strongly biological etiology for DD, and a dozen susceptibility genes have been suggested. Many of these genes point to common but previously unsuspected biological mechanisms, such as neuronal migration and cilia functions. I discuss here the state-of-the-art in genomic and neurobiological aspects of DD research, starting with short general background to its history.     

Pathogen virulence factors as molecular probes of basic plant cellular functions

To successfully colonize plants, pathogens have evolved a myriad of virulence factors that allow them to manipulate host cellular pathways in order to gain entry into, multiply and move within, and eventually exit the host for a new infection cycle. In the past few years, substantial progress has been made in characterizing the host targets of viral and bacterial virulence factors, providing unique insights into basic plant cellular processes such as gene silencing, vesicle trafficking, hormone signaling, and innate immunity. Identification of the host targets of additional pathogen virulence factors promises to continue shedding light on fundamental cellular mechanisms in plants, thus enhancing our understanding of plant signaling, metabolism, and cell biology.     

Protein-based tumor molecular imaging probes

Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging.     

Protein scaffold-based molecular probes for cancer molecular imaging

Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.