New putative phenol oxidase in ascidian blood cells.

The phenol oxidase system is ancient and ubiquitously distributed in all living organisms. In various groups it serves for the biosynthesis of pigments and neurotransmitters (dopamine), defence reactions and tissue hardening. Ascidians belong to subphylum Tunicata, which is considered the closest living relative to Vertebrates. Two phenol oxidases previously described for ascidians are vertebrate-like and arthropod-like phenol oxidases. In our present study, we described a new ascidian protein, Tuphoxin, with putative phenol oxidase function, which bears no sequence similarity with two enzymes described previously. The closest related proteins to Tuphoxin are mollusc haemocyanins. Unlike haemocyanins, which are oxygen transporting plasma proteins, Tuphoxin is synthesised in ascidian blood cells and secreted in the extracellular matrix of the tunic-ascidian outer coverings. Single mature transcript coding for this phenol oxidase can give several protein products of different sizes. Thus limited proteolysis of the initial protein is suggested. A unique feature of Tuphoxins and their homologues among Tunicata is the presence of thrombospondin first type repeats (TSP1) domain in their sequence which is supposed to provide interaction with extracellular matrix. The finding of TSP1 in the structure of phenol oxidases is new and we consider this to be an innovation of Tunicata evolutionary lineage.

Prognostic significance of the TNM system criteria, levels of serum insulin-like growth factors and their transport proteins, VEGF and MMP-7 in colorectal cancer.

The analysis of long-term results of treatment of 88 primary patients with colon adenocarcinoma at various stages of tumor process is presented, taking into account the TNM system criteria, and serum IGF-1, IGF-2, IGFBP-1, IGFBP-2, IGFBP-3, VEGF, and MMP-7 levels. The overall survival rate assessed by Kaplan-Meier method and Cox multivariate regression model was used as the criterion of prognosis. It was established that IGF-1, IGFBP-2 and VEGF serum levels along with the stage of colorectal cancer might be considered as statistically significant independent predictors of overall survival in patients.

Participation of PKG and PKA-related pathways in the IFN-γ induced modulation of the BKCa channel activity in human cardiac fibroblasts.

This study was devoted to elucidating the interferon (IFN)-γ-induced signaling pathway and the interaction between protein kinase G (PKG) and protein kinase A (PKA) through large-conductance Ca(2+)-activated K(+) channels in human cardiac fibroblasts. The IK currents were recorded using a whole-cell patch clamp method. A large depolarization (+50 mV) and a high Ca2+ concentration (pCa 6.0) were used in the internal pipette solution to activate only the KCa channels. Iberiotoxin (Ibtx), which selectively inhibits BKCa channels at a concentration of 100 nmol/l, caused a significant reduction of basal IK. Adding IFN-γ in the presence of Ibtx had no effect on IK. Application of the IFN-γ caused a significant reduction in total K+ current amplitude, recorded with a 500 ms depolarizing pulse duration, to +50 mV from a holding potential of -80 mV. We tested the involvement of the sGC/cGMP/PKG signaling pathway by using specific PKG inhibitor KT 5823, potent sGC inhibitor NS 2028, and specific sGC agonist BAY 41-8543. The obtained data confirmed that only sGC participated in the IFN-γ-mediated BKCa channel modulation, which was mediated further by PKA. This study represents first evidence about the participation of the IFN-γ in the mechanisms responsible for BKCa modulation in HCFs. We also believe that this process occurs via negative crosstalk between the PKG- and PKA-associated pathways.

Receptor Activator of Nuclear Transcription Factor NF-κB (RANK), Its Ligand RANKL, and Natural Inhibitor of RANKL Osteoprotegerin (OPG) in the Blood Serum of Patients with Primary Bone Tumors.

The content of components of the RANK/RANKL/OPG system, the key regulator of homeostasis in the bone tissue, in blood serum samples from 199 patients with primary bone neoplasms and 131 practically healthy volunteers was measured by ELISA. Borderline giantcell tumor of the bone with high osteoclastogenic and osteolytic activity is characterized by an increase in the level of all components of this system and highest ratio of sRANKL/OPG in the blood serum. Study indexes in patients with various benign neoplasms and tumor-like bone lesions were lower than in patients with giant-cell tumor. The patients with malignant bone tumors could be divided into 2 subgroups with opposite indexes of the RANK/RANKL/OPG system. The patients with osteosarcoma and Ewing sarcoma had a low level of sRANK, but a high level of sRANKL. The patients with chondrosarcoma and chordoma had a high level of sRANK, but a low level of sRANKL.

[Apoptin enhances the oncolytic activity of vaccinia virus].

Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.

Angiogenic factors in blood serum of bone tumor patients.

It is proven that angiogenesis and cartilage resorption processes involving vascular endothelial growth factor (VEGF) and its receptors play an important role in bone tissue regeneration and bone tumor development. A comparative analysis of VEGE VEGF-R1, VEGF-R2 and angiogenin content in blood serum of healthy persons, and patients with primary malignant, benign and borderline bone tumors was performed. 366 bone tumor patients aged 14-76 years were examined; 223 (60,9%) male and 143 (39,1%) -female. Control group included 48 practically healthy subjects aged 15-69 years; 25 (52,1%) male and 23 (47,9%) female. Serum VEGE VEGF-R1, VEGF-R2 and angiogenin concentrations measured using standard ELISA kits produced by "R&D Systems Inc. ", USA. It was shown that long-term treatment outcome (3-years overall survival) of typical osteosarcoma patients depended on tumor malignancy grade (determined by the criterion G). Baseline serum VEGF, its soluble receptors and angiogenin concentrations had no predictive value.

Matrix metalloproteinases 2, 7, 9 and tissue inhibitor of matrix metalloproteinase-1 in the sera of patients with bone tumors.

Comparative enzyme immunoassay of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitor-1 (TIMP-1) in the sera of 26 healthy individuals and 54 patients with primary osteal tumors before therapy revealed elevated TIMP-1 levels in the patients with classical central and periosteal osteosarcomas in comparison with the control. In patients, the level of MMP-9 significantly decreased compared to that in healthy individuals, while the levels of MMP-2 and MMP-7 remained unchanged. No differences in serum levels of MMP and TIMP-1 associated with gender, age, primary osteal tumor location and size were detected. Overall 3-year survival of patients with classical central osteosarcoma with serum level of MMP-9 below its median was higher than that of patients with MMP-9 level equal to above the median (90.9 ± 8.7 and 50.8 ± 23%, respectively).

[Study of coding sequences of variable regions in smallpox virus genome].

Potential ORFs were sought in the extended segments of terminal variable regions in the variola virus genome. These ORFs underwent a detailed structural and functional analysis and were compared both between themselves and with homologous ORFs of various orthopoxviruses. The most conservative and heterogeneous ORFs of 70 VARV strains were detected. The unique for VARV ORF (111 a.a) was revealed for Helder and Mary strains of the Russian collection. In addition, only in the Helder strain, ORF D14L was disintegrated into two separate ORFs. A number of ambiguities were found in the current databases for VARV ORF. The dominating type of evolution was ascertained to be stabilizing selection for analyzed ORF. It has been established that VARV ORF C3L, unlike other poxviral orthologs, undergoes an adaptive selection. These findings suggest that this gene plays an important role in human VARV adaptation.

[Differentiation of geographic biovariants of smallpox virus by PCR].

Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas.

Serum levels of endostatin, placental growth factor (PlGF), and fibroblast growth factors-1 and -2 (FGF-1 and FGF-2) were measured in 58 patients with primary osteosarcomas before therapy and in 21 healthy subjects. The incidence of serum FGF-1 in bone tumors was 2.5 times higher than in healthy individuals (p=0.004); significant levels of FGF-2, PlGF, and endostatin were detected in all examined subjects. The mean serum level of endostatin in healthy individuals was significantly lower than in the total group of patients with bone tumors (p=0.005). The level of FGF-1 in osteosarcomas was significantly higher than in chondrosarcomas (p<0.05). No appreciable differences in FGF-2 levels were detected in patients with tumors of different histological structure. The mean serum content of PlGF was virtually the same in healthy individuals and patients with bone tumors. A significant relationship between serum PlGF level and maximum tumor size (p=0.008) was detected in osteosarcoma. No relationships between the levels of FGF-1, FGF-2, PlGF, and endostatin were detected in healthy subjects and patients with primary tumors of the bones. Differences in 3-year overall survival values of patients with bone sarcomas with different initial serum levels of FGF-1 and endostatin were detected.

[Comparative analysis of variable regions in the genomes of variola virus].

Nucleotide sequences of two extended segments of the terminal variable regions in variola virus genome were determined. The size of the left segment was 13.5 kbp and of the right, 10.5 kbp. Totally, over 540 kbp were sequenced for 22 variola virus strains. The conducted phylogenetic analysis and the data published earlier allowed us to find the interrelations between 70 variola virus isolates, the character of their clustering, and the degree of intergroup and intragroup variations of the clusters of variola virus strains. The most polymorphic loci of the genome segments studied were determined. It was demonstrated that that these loci are localized to either noncoding genome regions or to the regions of destroyed open reading frames, characteristic of the ancestor virus. These loci are promising for development of the strategy for genotyping variola virus strains. Analysis of recombination using various methods demonstrated that, with the only exception, no statistically significant recombinational events in the genomes of variola virus strains studied were detectable.

[Immunogenic properties of recombinant vaccinia virus containing the human IL-2 gene].

A genetic construct of the human interleukin-2 (IL-2) gene within vaccinia virus (L-IVP strain) has been designed. The authors show the capacity of CV-1 cells infected with the recombinant vaccinia virus VV-SIL2 to secrete human IL-2 into the culture medium. Human IL-2 has been detected by immunoblotting. The sera from the animals immunized with the recombinant virus VV-SIL2 exhibited both human IL-2 and its antibodies throughout the observation period. This recombinant virus immunization induced both humoral and cell-mediated immune responses to human IL-2; the observed changes in the concentrations of cytokines are likely to suggest that the response predominantly followed a Th1 pathway. The study construct was nontoxic at the used concentrations and administration routes. The findings point that it is promising to investigate the adjuvant properties of the recombinant VV-SIL2 vaccine-based preparation for immunization in combination with various vaccines and to study this construct in therapy for cancer diseases.

[Construction of DNA-fragments' libraries with complete genomes of different variola strains].

Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.

Increase in serum concentration of FAS ligand as a possible mechanism for antithyroid cytotoxicity during autoimmune thyroid diseases.

We studied the dependence of serum cytotoxic activity on the contents of soluble apoptosis receptor and its soluble ligand in patients with autoimmune thyroid diseases.

[Comparative restriction enzyme analysis of the genome in variola virus strains from the Russian collection]. / Sravnitel'nyi restriktsionnyi analiz genomov shtammov virusa natural'noi ospy iz Rossiiskoi kollektsii.

Comparative RFLP analysis was for the first time performed for 21 variola virus (VARV) strains of the Russian collection with 20 amplicons covering the total VARV genome. The amplicons were synthesized in the long polymerase chain reaction. A database useful as a reference for identifying VARV strains was generated. VARV strains isolated in different geographical regions were compared and proved to vary mostly in variable genome regions. Each of the dendrograms constructed included three clusters of African, Asian, and VARV-alastrim isolates. The VARV-alastrim isolates differed to the greatest extent from the other strains. VARV strains isolated during an ecdemic variola burst in Moscow (1960) grouped with Asian isolates. Polymorphism of VARV strains was for the first time observed for a single variola burst with a few affected patients.

[The study of orthopoxvirus genes for Kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions]. / Izuchenie ortopoksvirusnykh genov, kodiruiushchikh Kelch-podobnye belki. II. Sozdanie variantov virusa ospy korov s napravlennymi deletsiiami genov.

Integrative plasmids p delta C, p delta D, and p delta G were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chicken embryos.

Tissue- and urokinase-type plasminogen activators and type 1 plasminogen activator inhibitor in melanomas and benign skin pigment neoplasms.

The content of urokinase- and tissue-type plasminogen activators and plasminogen activator inhibitor PAI-1 in the cytosol of primary and metastatic melanomas and benign skin pigment neoplasms was estimated by enzyme immunoassay. It was shown that local growth and invasion of melanomas are related to suppressed expression of tissue plasminogen activator. The content of urokinase plasminogen activator increases in patients with distant metastases and large thickness of the primary tumor.

[Expression of the apoptosis inhibitor soluble Fas antigen in patients with malignant and benign bone neoplasms]. / Ekspressiia ingibitora apoptoza--rastvorimogo Fas-antigena u bol'nykh so zlokachestvennymi i dobrokachestvennymi novoobrazovaniiami kostei.

This paper presents results of comparative enzyme immunoassay of soluble serum Fas antigen (sFas) in 98 patients with benign and malignant bone tumors, 15 patients with mechanical bone traumas, and 60 normal individuals of compatible age. The prevalence and concentration of sFas were significantly greater in patients with bone tumors than in normal donors and patients with mechanical bone traumas. The association of sFas with the pathogenesis of primary bone tumors is discussed in the paper.