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.

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.

[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.

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.

[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.

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.

[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.

[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.

High incidence of 550delA mutation of CAPN3 in LGMD2 patients from Russia.

Autosomal recessive limb gird muscular dystrophy (LGMD2) is a clinically and genetically heterogeneous group of diseases that are characterized by progressive atrophy and weakness of the proximal limb muscles. At least eight genetic loci leading to LGMD2 are recognized. The proportion of particular gene involved in producing different forms of LGMD2 shows a marked geographical variation. We studied 19 LGMD2 patients from Russia (15 families) and found calpain 3 (CAPN3) gene mutations in most of the patients studied. Sequence analysis of the fourth exons revealed two sibs - heterozygous compound for a 15-bp deletion (nt598-612) and 550 adenine deletion, and two sibs homozygous for a 550delA. We developed assay based on allele specific amplification (ASA) for rapid screening of the 550delA. The ASA assay of the LGMD2 patients under study showed that 7 patients from 6 families were homozygous for 550delA and 7 patients from 4 families were heterozygous for 550delA. A linkage analysis employing four microsatellites flanking the LGMD2A locus was performed. We found complete haplotype identity in most cases what favors the possibility of a common founder. Heterozygous carriers of 550delA were found in general population. The crude estimate of the mutation frequency is 1/150. Hum Mutat 15:295, 2000.

Internucleosomal chromatin degradation in myeloma and B-hybridoma cell cultures.

The activity of Ca/Mg-dependent endonuclease (CME) is strongly inhibited in myeloma X-63.Ag8.653 and B-hybridoma MLC-1c as compared with mouse splenocytes. Nevertheless, pronounced internucleosomal chromatin degradation occurs in both cell lines during long-term cultivation without passing. In isolated cell nuclei of X-63 the activation of CME, which precedes chromatin fragmentation in vivo and loss of cell viability, is revealed. The time-course of CME activation is opposite to cell proliferation and is not accompanied by alterations in enzyme quantity. The results suggest that cell death of X-63 and MLC-1c occurs via apoptosis, and involves the mechanisms controlling the activation and/or interaction of CME with chromatin.

[Search for new products of gene expression in human cardiac muscle. Microsequencing of proteins after two-dimensional electrophoresis]. / Poisk novykh produktov gennoi ékspressii v serdechnoi myshtse cheloveka. Mikrosekvenirovanie belkov posle dvumernogo élektroforeza.

Proteins of the human heart muscle were studied using modified two-dimensional electrophoresis. After separation, proteins were electroblotted onto Immobilon P membranes and several protein spots were used for microsequencing analysis. In most cases the proteins analyzed have blocked N-terminal amino acids. In order to study the primary structure of these proteins, hydrolysis in situ by trypsin followed by reversed-phase HPLC and microsequencing of the resulting peptides were performed. Four protein were identified in 8 analyzed fractions, specifically myosin light chain 1 (MLCl-V/sB), fatty-acid binding protein (heart isoform), alpha (B)-crystallin and alpha-tropomyosin. Amino acid sequences of two proteins were not found among human amino acid sequences collected in SWISSPROT bank (v. 21).

[DNA-methylase activity in human cardiac muscle. Association of DNA methylase activity with actin protein fractions]. / DNK-metilaznaia aktivnost' v serdechnoi myshtse cheloveka. Assotsiatsiia DNK-metilaznoi aktivnosti s fraktsiei aktinovykh belkov.

The column isoelectrofocusing activity of the nuclear extracts of the human cardiac muscle has revealed at pH 3.5-8.2 5 peaks of DNA-methylase. When one of these peaks (II) was analyzed by the two-dimensional gel electrophoresis 6 proteins (10, 25, 35, 43, 67 and 120 kDa) were separated. 43 kDa protein had electrophoretic properties similar to actins and was able to methylate cytosine in the DNA molecules. The comparative computer analysis of the primary structure of human actins and several bacterial DNA-methylases has shown the homology of the extensive fragments of these molecules.

[A new variant of myosin light chain 1 detected in human cardiac tissue]. / Novyi variant legkoi tsepi 1 miozina, obnaruzhennyi v serdechnoi myshtse cheloveka.

Proteins from biopsy of human heart muscle (n = 250) were studied. The supplementary fraction was found in a material from an individuum that coincided in molecular mass but differed in pI from the light chain of myosin usually expressed in the ventricular tissue of the heart muscle (LCM-1 v). The two-dimensional electrophoresis and immunoblotting have shown this supplementary fraction to be a rare allele of LCM-1 v.

[Human heart muscle proteins: two-dimensional electrophoretic analysis in embryos]. / Belki serdechnoi myshtsy cheloveka: dvukhmernyi élektroforeticheskianaliz u émbrionov.

Human myocardium proteins synthesized in the course of heart muscle development have been analyzed by two-dimensional electrophoresis. The quantitative change was found in representation of the main retractable proteins in the course of the heart muscle formation (the light myosin chains, tropomyosin, etc.). Four polymorphous variants of myocardium proteins were found, one of which is, possibly connected with the defect in heart development.

[Polymorphism of exon 4 in the CANP-3 gene in patients with primary myopathies]. / Polimorfizm ékzona 4 v gene CANP-3 u bol'nykh pervichnymi miopatiiami.

The structures of the gene for calpain (CANP-3) and of the DMD gene were analyzed in patients with primary myopathies [limb-girdle muscular distrophy (LGMD) and Duchenne-Becker myodystrophy (DBM)] from various regions of Russia. Via amplification of DNA isolated from the peripheral blood lymphocytes of 74 patients, extended deletions were found in 18 out of 55 patients with DBM. In none of the 19 patients with LGMD, were extended deletions in the CANP-3 gene found. In most patients with LGMD, the amplification of the promoter region and exons 1, 2, 3, 4, 5, and 6 of the CANP-3 gene yielded a single product of corresponding length, but in six patients (three sib pairs), amplification of exon 4 of the CANP-3 gene yielded two products of different size. The following single-strand conformation polymorphism (SSCP) analysis revealed a pronounced polymorphism of exon 4 of the CANP-3 gene in 14 out of 19 patients with LGMD. This structure of exon 4 of the CANP-3 gene was found neither in 16 patients with DBM who had deletions in the DMD gene nor in 16 patients with DBM who had no deletions in the DMD gene.

[Study of protein products of gene expression in cells with altered chromosome sets for genetic mapping]. / Izuchenie belkovykh produktov gennoi ékspressii v kletkakh s izmenennym naborom khromosom dlia geneticheskogo kartirovaniia.

Two-dimensional electrophoresis was used for analyzing proteins in hybrid cells that contained single human chromosomes (chromosome 5, chromosome 21, or chromosomes 5 and 21) against the background of the mouse genome. By comparing the protein patterns of hybrid and parent cells (about 1000 protein fractions for each kind of cell), five fractions among proteins of hybrid cells were supposedly identified as human proteins. The genes of two of them are probably located on chromosome 5, and those of other three, on chromosome 21. Moreover, analysis of proteins in fibroblasts of patients with the cri-du-chat syndrome (5p-) revealed a decrease in the content of two proteins, as compared with those in preparations of diploid fibroblasts. This fact was regarded as evidence that two corresponding genes are located on the short arm of chromosome 5. Methodological problems associated with the use of protein pattern analysis in cells with altered chromosome sets for the purposes of genetic mapping are discussed.