Laccase-Catalyzed Heterocoupling of Dihydroquercetin and p-Aminobenzoic Acid: Effect of the Reaction Product on Cultured Cells.

Derivatization of the natural flavonoid dihydroquercetin with p-aminobenzoic acid was carried out in an ethyl acetate/citric buffer biphasic system using laccase from the fungus Trametes hirsuta. The main reaction product yield was ~68 mol %. The product was characterized by 1H NMR, 13C NMR, and liquid chromatography-mass spectroscopy, and its structure was elucidated. The reaction product affected viability of cultured human rhabdomyosarcoma cells (RD cell line) in a dose-dependent manner and, therefore, can be of interest to pharmaceutical industry.

[The application of proteomic technologies for the analysis of muscle proteins of farm animals used in the meat industry (review)].

The review briefly summarizes the data on the development of proteomic technologies that became actively used in studies of the muscular proteins of farm animals used in the meat industry in 2006-2013. It has been noted that the main research trends are connected with the detection of changes in muscle proteins during post-mortem autolysis and the search for species-specific and other protein biomarkers. Particular publications regarding the development of methods based on proteomic technologies for monitoring the state of muscle proteins are considered. According to the analyzed data, we can conclude that the field is promising for the solution of a number of pressing problems in.applied biochemistry.

[Identification of the functional activity of synthetic polyamine analogues using a biotest system based on highly proliferating cultured human cells].

A new biotest system was developed based on highly proliferating human cell cultures (lines LNCaP and PC-3). With the help of this system, two known synthetic polyamines--alpha-difluoromethylornithine (DFMO) and methylglioxalbis(guanylhydrason) (MGBG)--as well as four new synthetic analogues difenyl containing amines (DFCA-1-DFCA-4) with molecular weights of 725.5 (DFCA-1), 755.5 (DFCA-2), 655.5 (DFCA-3), and 681.5 Da (DFCA-4) were tested. In this biotest system, DFMO (0.1-400 microM) did not reveal functional activity, whereas for MGBG a cytotoxic effect was registered (100-200 microM). DFCA-1, DFCA-2, and DFCA-4 had a similar effect at concentrations of 10 microM and higher; DFCA-3, at a concentration of 50 microM and higher. Thus, DFCA-1 has a higher level of antiproliferating activity and may be considered as the most potent cytostatic agent.

AGR2, ERp57/GRP58, and some other human protein disulfide isomerases.

This review considers the major features of human proteins AGR2 and ERp57/GRP58 and of other members of the protein disulfide isomerase (PDI) family. The ability of both AGR2 and ERp57/GRP58 to catalyze the formation of disulfide bonds in proteins is the parameter most important for assigning them to a PDI family. Moreover, these proteins and also other members of the PDI family have specific structural features (thioredoxin-like domains, special C-terminal motifs characteristic for proteins localized in the endoplasmic reticulum, etc.) that are necessary for their assignment to a PDI family. Data demonstrating the role of these two proteins in carcinogenesis are analyzed. Special attention is given to data indicating the presence of biomarker features in AGR2 and ERp57/GRP58. It is now thought that there is sufficient reason for studies of AGR2 and ERp57/GRP58 for possible use of these proteins in diagnosis of tumors. There are also prospects for studies on AGR2 and ERp57/GRP58 leading to developments in chemotherapy. Thus, we suppose that further studies on different members of the PDI family using modern postgenomic technologies will broaden current concepts about functions of these proteins, and this will be helpful for solution of urgent biomedical problems.

[Study of Dj-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients].

Two isoforms of Dj-1 protein were identified using a proteomic study in tissue specimens from two groups of patients with confirmed benign prostate hyperplasia (BPH) and prostate cancer (PCa). Dj-1 was also found in the cell lines PC-3, DU-145, LNCaP, BPH-1, and the lowest level of Dj-1 was found in BPH-1. Immunochemical study (ELISA) of serum levels of Dj-1, Bcl-2, IGF-1 and IGFBP-3 proteins revealed statistically significant distinctions between two groups of patients (p=0,004, Mann-Whitney test) only for Dj-1. Taken together, these data suggest that Dj-1 protein is a perspective biomarker candidate for PCa.

[A study of the single nucleotide polymorphism in seven genes (GHR, IGFBP3, IGFR1, IRS1, FMN1, ANXA2, TaGLN) in ethnic Russians and in patients with prostate cancer].

Using the RT-PCR method for allele discrimination, we examined nine known SNPs in seven genes (GHR, IGFBP3, IGFR1, IRS1, FMN1, ANXA2, TaGLN) in ethnic Russians and in patients with prostate cancer (PC). For Russian population data on genotype distribution in studied SNPs was obtained. It was revealed that six of nine analyzed sites in examined locus were polymorphic. Distributions of alleles and genotypes frequency of polymorphic site 1388 T/C (Leu463Pro) in gene FMN1 (rs2306277) were distinguished between patients and control groups (delta = 0.019; chi2 = 7.884). In particular, correlation of OO genotype with increased risk of PC was observed (OR = 2.1591 95% CI 1.2055-3.8726). Moreover, the analysis of the polymorphic site 2911G/A (Glu917Arg) in gene IRS1 (rs1801278) revealed the accumulation of allele A in cancer group in comparison with control group (chi2 = 4.038; p = 0.044). Thus, the obtained data indicate the possibility of participation of polymorphism in genes FMN1 and IRS1 in formation of predisposition to PC.

"Prostate cancer proteomics" database.

A database of Prostate Cancer Proteomics has been created by using the results of a proteomic study of human prostate carcinoma and benign hyperplasia tissues, and of some human-cultured cell lines (PCP, http://ef.inbi.ras.ru). PCP consists of 7 interrelated modules, each containing four levels of proteomic and biomedical data on the proteins in corresponding tissues or cells. The first data level, onto which each module is based, is a 2DE proteomic reference map where proteins separated by 2D electrophoresis, and subsequently identified by mass-spectrometry, are marked. The results of proteomic experiments form the second data level. The third level contains protein data from published articles and existing databases. The fourth level is formed with direct Internet links to the information on corresponding proteins in the NCBI and UniProt databases. PCP contains data on 359 proteins in total, including 17 potential biomarkers of prostate cancer, particularly AGR2, annexins, S100 proteins, PRO2675, and PRO2044. The database will be useful in a wide range of applications, including studies of molecular mechanisms of the aetiology and pathogenesis of prostate diseases, finding new diagnostic markers, etc.

Biochemical polymorphism of the growth hormone system proteins and its manifestations in human prostate cells.

The basic mechanisms are considered that are responsible for producing biochemical polymorphism of human proteins realized at three basic levels: the structures of genome and genes; the transcription and maturation of transcripts; the postsynthetic formation of functionally active protein products of gene expression. The data on biochemical polymorphism of growth hormone (GH) and some other proteins that are directly or indirectly necessary for its functioning and support this polymorphism by polylocus, polyallelism, alternative splicing, and various postsynthetic modifications are analyzed. The role of polymorphic proteins of the GH system is discussed in formation of a variety of oligomeric molecular structures of this system (multicomponent transport complexes, receptors, and endocellular protein ensembles involved in the regulation of gene expression). It is emphasized that such structural polymorphism significantly influences the biological effects in various parts of the GH system during physiological processes and in tumors, in particular in prostate cancer.

Proteomic analysis of human skeletal muscle (m. vastus lateralis) proteins: identification of 89 gene expression products.

Proteins from bioptates and autoptates of human skeletal muscle m. vastus lateralis were separated by O'Farrell two-dimensional gel electrophoresis (2DE). MALDI-TOF MS and MS/MS enabled identification of 89 protein spots as expression products of 55 genes. A modification of the O'Farrell's method including non-equilibrium electrophoresis in a pH gradient allowed detection--among major sarcomeric, mitochondrial, and cytosolic proteins--of several proteins, such as PDZ- and LIM domain-containing ones (pI > 8.70), fragments of known proteins, and a stable complex of heavy and light ferritin chains. The data underlie further studies of human skeletal muscle proteins in terms of molecular mechanisms of some physiological and pathological processes.

[A study of protein profile changes in differentiating human myoblasts].

The changes in the protein profile in cultured human myoblasts after induction of differentiation was studied by proteomic techniques (a combination of O'Farrell two-dimensional electrophoresis and subsequent protein identification by MALDI-TOF MS and MS/MS analyses). Forty-one proteins have been identified, 25 of which were present in both proliferating and differentiating myoblasts, which allows them to be considered as myoblast housekeeping proteins. The changes in the distribution of some isoforms of tropomyosins, S100 proteins, cofilin, etc. have been revealed. The possible role of these changes in the cell protein profile in the realization of the program of skeletal muscle cell differentiation is discussed.

[Clinicobiochemical study of diagnostic value of the panel including 10 potential protein markers of prostatic cancer].

We tested diagnostic value of the panel of 10 specially selected proteins--potential markers of prostatic cancer. A double blind method and proteomic technologies were used in complex clinicobiochemical examination of 20 patients with benign and malignant tumors. The same diagnosis were obtained by clinicomorphological criteria and protein markers in 13 (65%) cases. The highest diagnostic efficacy was achieved in prostatic cancer--11 cases (79%) vs 14 by clinicomorphological criteria.

[Determination of "amino acids conflicts" and amino acids substitutions in primary structures of 41 human proteins by use the proteomic technologies].

Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 of 300 identified proteins, in which the present of certain variants of amino acids ("conflicts") was recognized at several positions. Among the 93 registered amino acids "conflicts", seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Moreover, among prostate proteins the proteomic analysis revealed two isoforms of prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown, that proteomic technologies allow to specify effectively the features of primary structures and to characterize various kinds of polymorphism in many human proteins.

[Age-related changes of albumin and actin of human myocardium].

In the human myocardium tissue, proteomic technologies revealed the products of 2 genes (alpha-actin and albumin), existing as fragments; their appearance and increased contents correlated with age. The age-related variants differ from the mature forms by the absence of N-terminal fragments of the amino acid sequences. In the chronic ischemic heart disease (CIHD), these age-dependent proteins were found in 50% of cases (in the age group 31-40 years), while in the control group such combination was detected only in 10% of the examined individuals. Subsequent studies in this field would probably reveal molecular mechanisms responsible for impairment and/or ageing of the cardiac muscle and also of the adaptation-dysadaptations mechanisms in the CIHD.

[Identification of AGR2 protein, a novel potential cancer marker, using proteomics technologies].

A comparative analysis of the proteins in prostate tissues of the patients operated for hyperplasia (n = 7) or cancer (n = 5) was performed aiming to search for protein diagnostic markers. Differences in several minor proteins were detected using two-dimensional electrophoresis according to O'Farrel; among them, an additional protein with a molecular weight of 19 kDa and an isoelectric point of 9.0 was observed in four of the cancer cases. Mass spectrometry allowed this protein to be identified as the androgen-induced secreted protein AGR2. The possibility of using AGR2 as a diagnostic marker of prostate cancer is discussed.

Polymorphism of delta3,5-delta2,4-dienoyl-coenzyme A isomerase (the ECH1 gene product protein) in human striated muscle tissue.

Two polymorphic variants of the ECH1 gene product protein (delta3,5-delta2,4-dienoyl-coenzyme A isomerase) have been revealed by proteomics methods in samples of human striated muscle tissue. These variants are identical in molecular weight (29.7 kD) but different in pI values (6.57 and 6.75) and in amino acid substitution (41 E-->A) confirmed by mass spectrometry. The same type of polymorphism has been detected in samples of different tissues of the same person, so these variants are considered (also based on other data) to be allelic. The rates of these alleles in two representative cohorts of Moscow and Minsk residents are similar.

[New approaches to molecular diagnosis of prostatic cancer].

We compared prostatic proteins in patients operated for adenoma or cancer. 630 protein fractions were obtained from each tissue sample after fractionation by two-dimentional electrophoresis according to O'Farrell. Comparison of the samples from adenoma and cancer showed their difference by 7 proteins among which were isoforms of glyceraldehydes-3-phosphate-dehydrogenase, alpha-collagen and several little known proteins. Most of the cancer patients had the protein with molecular mass 19 kDa and isoelectric point 9.0. By the results of mass-spectrometry this protein was identified as androgen-induced secreted protein AGR2. This protein is considered a potential oncomarker. Prospects of some postgenome technologies for detection of new diagnostic markers of prostatic cancer are discussed.

[NP-detecting DNA technologies: solving problems of applied biochemistry].

Heterozygosity of CANP3, ACTN3, and GHR genes in specialized collections was studied using state-of-the-art DNA technologies for DNA analysis. A new dinucleotide deletion (AC) at the beginning of exon 21 was identified in five individuals with heterozygous CANP3 gene. Analysis of polymorphism (SNP1747 C-->T) of ACTN3 gene demonstrated a positive association of allele C with a high muscular performance. Real-time PCR assay of SNP1630 (A-->C) in GHR gene suggested a putative negative association of allele C of this SNP with a high muscular performance.

[Effects of myostatin and other growth factors on cultured human cells]. / Vliianie miostatina i nekotorykh rostovykh faktorov na kul'tiviruemye kletki cheloveka.

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.

Proteomic studies of human and other vertebrate muscle proteins.

This review summarizes results of some systemic studies of muscle proteins of humans and some other vertebrates. The studies, started after introduction of two-dimensional gel electrophoresis of O'Farrell, were significantly extended during development of proteomics, a special branch of functional genomics. Special attention is paid to analysis of characteristic features of strategy for practical realization of the systemic approach during three main stages of these studies: pre-genomic, genomic (with organizational registration of proteomics), and post-genomic characterized by active use of structural genomics data. Proteomic technologies play an important role in detection of changes in isoforms of various muscle proteins (myosins, troponins, etc.). These changes possibly reflecting tissue specificity of gene expression may underline functional state of muscle tissues under normal and pathological conditions, and such proteomic analysis is now used in various fields of medicine.

Comparison of cytotoxicity of aminoglycoside antibiotics using a panel cellular biotest system.

The cytotoxicity of four aminoglycoside antibiotics was studied by estimation of the dose-effect relationship using a panel cellular biotest system including cell cultures for test objects. The cultures represented 4 differentiation types: normal human fibroblasts and myoblasts, human or Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells. It was found that three widely used antibiotics gentamicin, kanamycin, and neomycin exhibit similar, but not identical cytotoxicity parameters and differ distinctly from geneticin. Hence, the proposed panel biotest system helps to quantitatively evaluate and differentiate the effects of bioactive substances with similar chemical structure.