[Single-Domain Antibodies Used to Pretreat the Human Urinary Proteome in Cancer Biomarker Testing].

A number of single-domain antibodies (nanobodies) obtained previously to major marker blood proteins were tested as tools to preprocess urine samples from patients with bladder cancer. Nanobody-based tools demonstrated unique possibilities for noninvasive diagnostic studies along with other conventional methods, such as electrophoresis and, in prospect, mass spectrometric analysis. A testing of 22 samples from bladder cancer patients showed that the development of bladder cancer is accompanied by an increase in the urine contents of major blood proteins, including those known as potential bladder cancer biomarkers. New nanobody-based immunosorbents allow both specific enrichment and specific removal of particular antigenic proteins and subproteomes associated with them from a biological fluid. The isolation of immune complexes from the urine of a particular patient is of particular interest. An initial study of the complexes showed not only increased contents of IgA and IgG at advanced stages of the disease, but also many other components, which provide potential biomarkers of the pathological process in a particular patient. It is intended to use the approaches proposed in this work in a future larger-scale study of urine samples from patients with bladder cancer at different stages of the disease in order to identify new promising biomarkers of bladder cancer.

[Prospective Applications of Single-Domain Antibodies in Biomedicine].

Methods that utilize highly specific antibodies, anti-idiotypic antibodies, various recombinant molecules with antibody properties and immunocorrection and immunoprophylaxis with the help of vaccines are in demand and are intensely developed in the field of biomedicine. Techniques to generate specific single-domain recombinant antibodies (nanobodies) and their derivatives have raised great expectations in the past years. The review considers the recent literature data on the use of nanobodies in basic research, diagnosis, and design of new immunotherapeutic agents. Special sections focus on the prospects of using nanobodies as targeted molecules of microbiota components, the use of anti-idiotypic nanobodies, and a search for promising targets for early diagnosis based on nanobodies.

[Differentially Expressed Long Noncoding RNAs in the Promoter Region of the fork head Gene in Drosophila melanogaster Detected by Northern Blot Hybridization].

It is known that long (200-300 nucleotides and longer) non-protein-coding RNAs (ncRNAs) tissue-specifically expressed from the regulatory regions of developmental genes can regulate the transcription of the mRNA of these genes. In this study, an attempt is made to identify differentially expressed ncRNAs in the extended promoter region of the fork head (fkh) gene of the fruit fly Drosophila melanogaster. We investigated four preparations of the total RNA: from embryos, from adult flies (separately from females and males), and from the S2 cell line of cultured Drosophila cells. In the total RNA preparations from embryos and adult flies, the levels of fkh expression differed substantially, whereas in S2 cells its expression is not detected at all (shown in this work). We perform classical Northern blot analysis of gel-separated RNAs hybridized to a series of radioactively labeled DNA fragments corresponding to the adjacent and partially overlapping regions of the promoter region of the fkh gene. Several previously unknown differentially expressed ncRNAs are detected, including those in the regions overlapping with the previously detected regulatory elements (TRE1 and salivary gland enhancer sgE) and the transcription start site of the fkh gene. The collected data complement and clarify the results of the previously conducted RNA-seq experiments, in particular, in terms of the length of the detected RNAs. These results may serve as a foundation for further studies of the mechanisms of tissue-specific regulation of the fkh gene expression.

[A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma].

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.

Molecular analysis of heavy chain-only antibodies of Camelus bactrianus.

In this work, IgG content and structures of antigen-binding domains and hinge regions of different IgG subtypes of Camelus bactrianus were analyzed in detail for the first time. Our data demonstrate that C. bactrianus contains a very large amount of heavy chain-only antibodies that can be used as a source of VHH domain-containing molecules. Despite some minor sequence differences identified in this study, C. bactrianus VHH domains possess principally the same unique features as those of C. dromedarius and the llama. These features are important for developing an efficient phage display-based antibody selection technology. We conclude that C. bactrianus is a very suitable animal to raise an immune response that serves as a source to identify antigen-specific VHHs selected after phage display.

Experimental models of arthritis in which pathogenesis is dependent on TNF expression.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint damage as well as systemic manifestations. The exact cause of RA is not known. Both genetic and environmental factors are believed to contribute to the development of this disease. Increased expression of tumor necrosis factor (TNF) has been implicated in the pathogenesis of RA. Currently, the use of anti-TNF drugs is one of the most effective strategies for the treatment of RA, although therapeutic response is not observed in all patients. Furthermore, due to non-redundant protective functions of TNF, systemic anti-TNF therapy is often associated with unwanted side effects such as increased frequency of infectious diseases. Development of experimental models of arthritis in mice is necessary for studies on the mechanisms of pathogenesis of this disease and can be useful for comparative evaluation of various anti-TNF drugs. Here we provide an overview of the field and present our own data with two experimental models of autoimmune arthritis - collagen-induced arthritis and antibody-induced arthritis in C57Bl/6 and BALB/c mice, as well as in tnf-humanized mice generated on C57Bl/6 background. We show that TNF-deficient mice are resistant to the development of collagen-induced arthritis, and the use of anti-TNF therapy significantly reduces the disease symptoms. We also generated and evaluated a fluorescent detector of TNF overexpression in vivo. Overall, we have developed an experimental platform for studying the mechanisms of action of existing and newly developed anti-TNF drugs for the treatment of rheumatoid arthritis.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165).

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A(165) has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A(165). We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A(165) as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A(165), which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A(165)-dependent pathological neoangiogenesis.

High-Affinity Single-Domain Antibodies for Analyzing Human Apo- and Holo-Transferrin.

A highly efficient technology for generating new monoclonal single-domain recombinant antibodies (nanobodies) was used to obtain a panel of nanobodies recognizing human apo- and/or holo-transferrin. This article is devoted to the primary analysis of the properties of two different variants of the new nanobodies obtained by us, as well as to the demonstration of the unique potential of their application for diagnostic studies. The simultaneous use of immunosorbents based on these nanobodies apparently makes it possible to detect changes in the relative abundance of apo- and holo-transferrin in human biological fluids. Such changes could potentially be indicative of an increased risk or degree of development of pathological processes, such as malignant neoplasms in humans.

["Camel nanoantibody" is an efficient tool for research, diagnostics and therapy].

This short review provides an introduction to the rapidly developing field of generation and utilization of "camel nanoantibodies" (or "nanobodies"). The term "nanoantibody" or "nanobody" was given to single-domain variable fragments of special type of antibodies that naturally exist (in addition to classical types of antibodies) in blood of Camelidae family animals and in some chondrichthyan fishes. The existence of very efficient technology of nanobody generation and some very useful characteristic features promise a big potential for their use in immunobiotechnology and medicine.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene.

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators, which is required to maintain active states of Hox gene expression during development. We have purified from Drosophila embryos a trithorax acetylation complex (TAC1) that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression.

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each approximately 400 bp) contain both TRX- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (approximately 90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group gene pleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators.

Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

[Immunoprecipitation mapping of the TRX-associated chromosome elements in the fork head gene promoter in the Drosophila melanogaster salivary gland cells].

Using the method of immunoprecipitation of the in vivo crosslinked and sheared by sonication chromatin, mapping of potential trithorax-associated regulatory elements within the extended (9 kb) promoter region of the fork head gene (fkh) in the Drosophila melanogaster salivary gland cells was performed. Relative homogeneity of the salivary gland cells, along with the parallel use of the antibodies to different domains of the same trithorax protein (TRX), and the introduction of cross-hybridization steps for additional specific enrichment of initial DNA libraries, provided improvement of the method effectiveness and identification of one major and two less expressed potential TRX-binding sites.

[Single-domain adapted antibodies against Chlamydia trachomatis, preserving the development of chlamidic infection in vitro]. / Odnodomennye adaptirovannye antitela protiv Chlamydia trachomatis, podavliaiushchie razvitie khlamidiinoi infektsii v usloviiakh in vitro.

The technology for the generating of single-domain recombinant monoclonal antibodies (nanoantibodies) based on the immunization of a camel, cloning of induced sequences encoding single-domain antigen-recognizing fragments of non-canonical camel antibodies, as well as functional selection of clones of nanoantibodies by the phage display method, was used to obtain new effective tools for more efficient diagnostics of Chlamydia infection and to develop new approaches for effective therapy. Two promising nanoantibodies were obtained. They showed effective binding to extracellular and intracellular forms of C. trachomatis, and also had activity that inhibited the development of chlamydial infection in vitro.

Biosynthesis and cytoplasmic distribution of small poly(A)-containing B2 RNA.

Previously, we described a small polyadenylated RNA predominantly located in cytoplasm and hybridizing with the ubiquitous B2 sequence of the mouse genome (Kramerov, D.A., Lekakh, I.V., Samarina, O.P. and Ryskov, A.P. (1982) Nucleic Acids Res. 10, 7477-7491). This 180-300 nucleotide long RNA was designated B2 RNA. Here, we demonstrate that B2 RNA is complementary to just one of the strands of cloned B2 sequence. The synthesis of B2 is rather resistant to ultraviolet irradiation of Ehrlich ascites carcinoma cells. The treatment of the cells with alpha-amanitin at a concentration completely blocking the formation of small nuclear RNAs U1, U2 and U3 does not interfere with the B2 RNA synthesis. These results suggest that B2 RNA formation is directly transcribed with the aid of RNA polymerase III, rather than being formed in the course of the processing of large RNA molecules which are known to contain a lot of B2 sequences. We also surprisingly found that the synthesis of up to 50% of long poly(A) +RNA in Ehrlich carcinoma cells is rather resistant to alpha-amanitin. The possible role of genetic elements including B2 sequences able to promote large RNA-polymerase III transcripts is discussed. B2 RNA in the cytoplasm is incorporated into the ribonucleoprotein particles, both small (12-18 S) and heavy. The latter probably correspond to informosomes. After deproteinization of heavy particles, a major part of B2 RNA still cosediments with mRNA and is split from it only after denaturation. We suggest that the B2 RNA of heavy ribonucleoproteins is associated with mRNA by short complementary stretches. About half of the B2 RNA is recovered in the cytoskeletal fraction. The possible role of B2 RNA in mRNA transport or in translation regulation is discussed.

Conservation of structure and expression of the trithorax gene between Drosophila virilis and Drosophila melanogaster.

The Drosophila melanogaster trithorax gene encodes several large RNAs which are expressed in complex patterns in the embryo. The D. virilis trithorax gene was isolated and sequenced. It produces a similar to D. melanogaster set of transcripts, and it encodes a protein that shows sequence similarity in several domains which are also conserved in human homologue, ALL-1/HRX. Previous experiments have suggested that a distinct expression domain of trithorax in the posterior region of the embryo is required to maintain expression of the BX-C genes (Sedkov et al., 1994, Development 120, 1907-1917). At cellular blastoderm, trithorax RNA expression in D. virilis embryos is also confined to the posterior portion of the presumptive mesoderm. This finding supports the idea that the specific BX-C-related expression domain is an essential feature of the trithorax gene.

Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein.

The products of the trithorax and Polycomb groups genes maintain the activity and silence, respectively, of many developmental genes including genes of the homeotic complexes. This transcriptional regulation is likely to involve modification of chromatin structure. Here, we report the cloning and characterization of a new gene, trithorax-related (trr), which shares sequence similarities with members of both the trithorax and Polycomb groups. The trr transcript is 9.6 kb in length and is present throughout development. The TRR protein, as predicted from the nucleotide sequence of the open reading frame, is 2431 amino acids in length and contains a PHD finger-like domain and a SET domain, two highly conserved protein motifs found in several trithorax and Polycomb group proteins, and in modifiers of position effect variegation. TRR is most similar in sequence to the human ALR protein, suggesting that trr is a Drosophila homologue of the ALR. TRR is also highly homologous to Drosophila TRITHORAX protein and to its human homologue, ALL-1/HRX. However, preliminary genetic analysis of a trr null allele suggests that TRR protein may not be involved in regulation of homeotic genes (i.e. not a member of the trithorax or Polycomb groups) or in position effect variegation.

Advances in the analysis of DNA sequence variations using oligonucleotide microchip technology.

The analysis of DNA variation (polymorphisms and mutations) on a genome-wide scale is becoming both increasingly important and technically challenging. An integration of a growing number of molecular biological methods of DNA-sequence analysis with the high-throughput feature of oligonucleotide microarray-based technologies is one of the most promising current directions of research and development.