Vol 1, No 3 (2009)

This section “Forum” is about the pharmaceutical industry in Russia. We were encouraged to debate this topic after the unveiling of the Strategy of Development of the Pharmaceutical Industry in the Russian Federation developed by the Ministry of Industry and Trade of the Russian Federation. The majority of our experts, who are authorities in the federal government, business, academia, and industrial science, believe that Russia needs a fully developed pharmaceutical industry. What are the main arguments for an intensive development of a Russian pharmaceutical industry- In our opinion, there are four major reasons.

The share of locally produced insulin on the Russian market estimated at more than seven billion roubles in 2008 is, in reality, microscopic: national industry covers only 2 % of the market (see Table 1). This is unreasonably small, especially because а) Russian producers have enough capacity to produce insulin for the entire country, with similar or even higher quality as compared with foreign products, b) hundreds of thousands of insulindependent patients in some particular political or economical circumstances could suffer without this life-saving treatment. The World Health Organization recommends that any country with a population of more than 50 millions have its own manufacturing base for insulin. Anatoly Miroshnikov is an assistant director of the M.M. Shemiakin and Y.A. Ovchinnikov’ Institute of Bioorganic Chemistry of the RAS, which produces insulin with the trade name Insuran. He talks to Acta Naturae on why Russia needs its own factories for the production of genetically engineered drugs.

This paper reviews the chemical and functional aspects of the posttranslational modifications of proteins, which are achieved by the addition of various groups to the side chain of the amino acid residue backbone of proteins. It describes the main prosthetic groups and the interaction of these groups and the apoenzyme in the process of catalysis, using pyridoxal catalysis as an example. Much attention is paid to the role of posttranslational modification of proteins in the regulation of biochemical processes in live organisms, and especially to the role of protein kinases and their respective phosphotases. Methylation and acetylation reactions and their role in the “histone code,” which regulates genome expression on the transcription level, are also reviewed. This paper also describes the modification of proteins by large hydrophobic residues and their role in the function of membrane-associated proteins. Much attention is paid to the glycosylation of proteins, which leads to the formation of glycoproteins. We also describe the main non-enzymatic protein modifications such as glycation, homocysteination, and desamidation of amide residues in dibasic acids.

Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in “postmortem” mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems.. Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.

In this work we describe an unusual enzyme from Leishmania major (Arabinokinase/Pyrophosphorylase) that catalyzes the synthesis of GDP-Darabinopyranose (GDP-D-Ara p) via a D-arabinose-1-phosphate intermediate in the presence of ATP and GTP. Our data indicate GDP-D-Ara p transport in vivo by the LPG2 multispecific nucleotide sugar transporter into the Leishmania Golgi apparatus, in which it can be used by glycosyltransferases as a donor substrate for glycosylation.

The crystal structure of the ternary complex of NAD--dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. С -1 with the cofactor (NAD-) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacements parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the temperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreement with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction.

The toxic and genotoxic effects of silver nanoparticles were studied on injected mice (BALB/c line) in vivo. A water solution of silver nanoparticles (SNP) with particle sizes of 9±6 nm was obtained by means of the original method of biochemical synthesis. The effect of the SNP solution was compared to those of AOT (anionic surfactant used as SNP stabilizer) and silver nitrate (i.e. Ag- ions) introduced as water solutions. In studies of the toxic effects, the death of mice was registered 12-24 hours after injection only at two maximum dozes of SNP (equivalent to 0.54 and 0.36 gAg/l). It is shown that the toxic effect decreases in the sequence SNP>AOT>>AgNO3. The LE50/30 values for SNP and AOT are equal to 0.30±0.07 gAg/l and 13.3±2.1 gAg/l, respectively. Genotoxic effects were assessed by the abnormal sperm heads test and neutral Comet assay. The frequencies of abnormal sperm heads (ASHs) did not differ after treatment by SNP and AOT, but both were significantly higher than those found with AgNO3 and in control mice. Comet assay showed an increase of the DNA percentage in the comet tail in spleen cells after the injection of SNP and AOT in concentrations of ½ LE50/30. Tail DNA % was 32.8±1.3 and 26.3±1.7%, respectively, vs 16.2±0.7% for the untreated control. To sum up, these tests showed that the genotoxic effects of the SNP solution are associated with the presence of AOT rather than SNP.

Cell regulation of Ph +cell proliferation and differentiation has been studied ex vivo in various chronic myeloid leukemia (CML) patients. The regulation is provided by alternation of effective stages of proliferation and maturation with inhibition of Ph+ cell proliferation by accumulating neutrophils under apoptosis blockage. The alternation of stages consists of switching stage 1 (effective proliferation) to stage 2 (effective maturation) and proceeds according to the 1/2 -1/2/1 or 2/1-2/1/2/1 schemes. The kinetic plots of alternations pass through control points of crossing plots, where the parameters of proliferation and maturation are equal. The indices of P/D efficiency (ratio of proliferation and maturation rates) are 1.06±0.23 and don’t depend on time, alternation order, or sources of Ph+ cells – CML patients. During stages alternation, conversely, the parameters of Ph + cell proliferation and maturation vary. The proliferation stages are characterized by increased proliferating cells content, a decreased number of neutrophils, and apoptosis induction. At the maturation stages, conversely, apoptosis is inhibited, the number of mature neutrophils increases, while immature Ph + cells decrease. High content neutrophils inhibit the proliferation of Ph + cells and impair their own maturation by inversion of maturation order, probably through a feedback mechanism. The regulation differences ex vivo reveal three types of Ph + cells from various individual CML patients, distinguished by the number and duration of alternating stages of proliferation and maturation. Ph + cells types 1 and 2 have one prolonged stage of effective proliferation or effective maturation with efficiency indices P/D 1 = 1-20 or P/D 2 ≤ 1. At the same time period, the proliferation and differentiation of the Ph + cells type 3 proceeds with repeated alternations of stages with P/D 1 = 1-4 or P/D 2 ≤ 1. Type 1 Ph + cells (~20%) were isolated from patients in advanced stages of CML, while Ph + cells types 2 and 3 (30 and 50% correspondingly) were isolated from CML chronic phase patients sensitive to chemotherapy.

Psoriasis was used as a model to analyze the pathogenetic pathways of immune-mediated inflammatory diseases, and the results of bioinformatic, molecular-genetic and proteomic studies are provided. Cell mechanisms, common for the pathogenesis of psoriasis, as well as Crohn’s disease, are identified. New approaches for immune-mediated diseases are discussed.