Solid phase extraction of PCDDs/PCDFs and dioxin-like PCBs from edible oils and fats.

We have studied the feasibility of solid phase extraction of PCDDs/PCDFs and dioxin-like PCBs from oils and fats as the first step of a sample preparation procedure that would not involve the chemical decomposition of the matrix. A few experimental setups using dual-layer columns packed with various brands of active carbon were tested. The use of a dual-layer microcolumn with AX-21 and Carboxen 1000 carbons for dioxins extraction from animal fats, vegetable oils and powdered milk gave satisfactory recoveries which met the European Union Commission Regulation 2017/644 criteria for dioxin analysis. The developed method of solid phase extraction on a dual-layer carbon column requires lower amounts of solvents and sorbents, tolerates high amounts of fat and can be used in both manual and automated sample preparation procedures. The recoveries obtained for the most toxic congeners (2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF) are within 79-119%.

Preparation and functional evaluation of RGD-modified streptavidin targeting to integrin-expressing melanoma cells.

The vertical growth stage is the most dangerous stage of melanoma and is often associated with a poor prognosis. The increased invasiveness and metastasis that is typical for vertically growing melanoma are mediated by the molecules of cell adhesion (particularly, integrins). Integrin αvß3, which is abundantly expressed on melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for melanoma diagnostics and therapy. Integrin αvß3 is known to recognize the arginine-glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural ligands. Here expression vectors bearing the genes of fusion proteins have been constructed for producing these proteins in Escherichia coli. Such fusion proteins consist of a peptidic 'address,' targeting the integrins on melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric protein, namely, streptavidin, that is able to achieve high-affinity binding of d-biotin (K(d) = 10(-15) M) and confer avidity to the address peptide. This binding ability facilitates the generation of anti-melanoma diagnostic and therapeutic agents using the appropriate biotin derivatives. These recombinant proteins were purified from the periplasm of E.coli using columns with 2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human melanoma cells.

RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies.

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G1/S boundary.

Molecular epidemiology of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 strains from Russian patients with classic, posttransplant, and AIDS-associated Kaposi's sarcoma.

We report the molecular characterization of 38 new Kaposi's sarcoma-associated herpesvirus (KSHV) strains from Russian patients with either classic (25 cases), epidemic/AIDS-associated (7 cases), or posttransplant/immunosuppressed patients (6 cases), or Kaposi's sarcoma (KS). While a complete sequence of the K1 gene (870 bp) was obtained from 30 strains, only partial sequences of the hypervariable regions VR1 (372 bp) and/or VR2 (381 bp) of the K1 gene were obtained from eight strains of KS paraffin blocks. Sequence comparison and phylogenetic studies indicate that the novel KSHV strains belong to either the A subtype (28 cases) or the C subtype (10 cases). Within the 28 strains of A subtype, 24 (86%) belong to the large A' subgroup, mostly A1 and A1' clades, and 4 belong to the A" subgroup, mostly A3 clade. Within the 10 strains of subtype C, 4 were of C' subgroup, and 6 of the C". Some molecular variants of subtype A' were observed, with 3 strains exhibiting an insertion of a single amino acid at the position 65 and 2 strains (both from AIDS-KS) with an unique deletion of 17 amino acids in the VR2 region. Polymerase chain reaction-based subtyping of the K14.1 genomic region indicated that most (23/32) of the novel strains belonged to the P subtype. The results indicate that despite a wide genetic diversity of A and C K1 subtypes of KSHV strains present in Russia, most are closely related and belong to the A1 or A1' molecular clades suggesting a common origin. This study also expands the data regarding the absence of any correlation between a K1 molecular subtype and a specific KS type (classic, epidemic, or posttransplant), as well as between the K1 and K14.1 molecular subtypes.