Biochemistry (Moscow) (v.79, #6)

Circahoralian (Ultradian) metabolic rhythms by V. Y. Brodsky (483-495).
This review presents data concerning metabolic rhythms with periods close to one hour (20 to 120 min): their occurrence, biochemical organization, nature, and significance for adaptations and age-related changes of cells and organs. Circahoralian (ultradian) rhythms have been detected for cell mass and size, protein synthesis, enzyme activities, concentration of ATP and hormones, cell respiration, and cytoplasm pH. Rhythms have been observed in bacteria, yeasts, and protozoa, as well as in many cells of metazoans, including mammals, in vivo and in cell cultures. In cell populations, the rhythms are organized by direct cell-cell communication. The biochemical mechanism involves membrane signal factors and cytoplasmic processes resulting in synchronization of individual oscillations to a common rhythm. Phosphorylation of proteins is the key process of coordination of protein synthesis and enzyme activity kinetics. The fractal nature of circahoralian rhythms is discussed as well as the involvement of these rhythms in adaptations of the cells and organs. Senescent decrease in rhythm amplitudes and correspondingly in cell-cell communication has been observed. The possibility of remodeling these changes through the intercellular medium has been predicted and experimentally shown. Perspectives for studies of the organizers and disorganizers of cell-cell communication in the intercellular medium along with appropriate receptors are discussed with special emphasis on aging and pathology. One perspective can be more precise definition of the range of normal biochemical and physiological state with the goal of correction of cellular functions.
Keywords: metabolic rhythms; kinetics of protein synthesis; kinetics of enzyme activities; direct cell-cell communication; fractals; aging

To perform their functions, many DNA-dependent proteins have to quickly locate specific targets against the vast excess of nonspecific DNA. Although this problem was first formulated over 40 years ago, the mechanism of such search remains one of the unsolved fundamental problems in the field of protein-DNA interactions. Several complementary mechanisms have been suggested: sliding, based on one-dimensional random diffusion along the DNA contour; hopping, in which the protein “jumps” between the closely located DNA fragments; macroscopic association-dissociation of the protein-DNA complex; and intersegmental transfer. This review covers the modern state of the problem of target DNA search, theoretical descriptions, and methods of research at the macroscopic (molecule ensembles) and microscopic (individual molecules) levels. Almost all studied DNA-dependent proteins search for specific targets by combined three-dimensional diffusion and one-dimensional diffusion along the DNA contour.
Keywords: protein-DNA interactions; DNA target search; one-dimensional diffusion; DNA repair; restriction endonucleases; transcription factors

Mitochondrial energy-dissipating systems (alternative oxidase, uncoupling proteins, and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings by O. I. Grabelnych; O. A. Borovik; E. L. Tauson; T. P. Pobezhimova; A. I. Katyshev; N. S. Pavlovskaya; N. A. Koroleva; I. V. Lyubushkina; V. Yu. Bashmakov; V. N. Popov; G. B. Borovskii; V. K. Voinikov (506-519).
Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (−2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.
Keywords: Triticum aestivum L.; cold hardening; reactive oxygen species; gene expression; energy-dissipating systems of mitochondria

The size of the light-harvesting antenna of higher plant photosystem ii is regulated by illumination intensity through transcription of antenna protein genes by M. M. Borisova-Mubarakshina; D. V. Vetoshkina; N. N. Rudenko; G. N. Shirshikova; T. P. Fedorchuk; I. A. Naydov; B. N. Ivanov (520-523).
In arabidopsis plants, with an increase in illumination intensity during growth the extent of reduction of the plastoquinone pool in the photosynthetic electron transport chain increased, whereas the effective quantum yield of photosynthesis decreased. After 5 days of growth under high illumination intensity, these parameters in high light returned to values observed in “shade-adapted” plants in low light. During the same period, the size of the antenna decreased, correlating with a decrease in the amounts of proteins of peripheral pigment-protein complexes. It was found that the decrease in the amounts of these proteins occurred due to suppression of transcription of their genes.
Keywords: photosynthesis; adaptation to illumination intensity; plastoquinone pool; light harvesting antenna; gene expression

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.
Keywords: mesenchymal stem cells; Wnt/β-catenin signaling pathway

A unique disulfide bridge of the thermophilic xylanase syxyn11 plays a key role in its thermostability by X. Yin; Y. Yao; M. C. Wu; T. D. Zhu; Y. Zeng; Q. F. Pang (531-537).
Based on the hyperthermostable family 11 xylanase (EvXyn11TS) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5–Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11C5T-encoding gene, Syxyn11 C5T, was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11C5T, were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11C5T decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.
Keywords: xylanase; thermostability; disulfide bridge; computational prediction; site-directed mutagenesis

HMGA1 is a new target of miR-195 involving isoprenaline-induced cardiomyocyte hypertrophy by Xiang-Yu You; Jiong-Hua Huang; Bin Liu; Shao-Jun Liu; Yun Zhong; Shi-Ming Liu (538-544).
Emerging data have shown that microRNAs (miRNAs) have important functions in the processes of cardiac hypertrophy and heart failure that occur during the postnatal period. Cardiac overexpression of miR-195 results in pathological cardiac growth and heart failure in transgenic mice. In the present study, we analyzed the roles of miR-195 in cardiomyocyte hypertrophy and found that miR-195 was greatly upregulated during isoprenaline-induced cardiomyocyte hypertrophy. By using mRNA microarray and molecular approach, we identified a novel putative target of miR-195 called high-mobility group A1 (HMGA1). Total mRNA microarray showed that HMGA1 was downregulated in primary cardiomyocytes that overexpressed miR-195. Using luciferase activity assay, we demonstrated that miR-195 interacts with the 3′-untranslated region of HMGA1 mRNA. Moreover, we showed that miR-195 in primary cardiomyocytes downregulates the expression of HMGA1 at the protein level. Taken together, our data demonstrated that miR-195 can negatively regulate a new target, HMGA1, which is involved in cardiomyocyte hypertrophy.
Keywords: microRNA-195; cardiomyocyte hypertrophy; microRNA target; HMGA1

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the inter-action of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E ≫ K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.
Keywords: nucleotide excision repair; XPA mutants; DNA binding

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria by Yu. L. Baburina; A. E. Gordeeva; D. A. Moshkov; O. V. Krestinina; A. A. Azarashvili; I. V. Odinokova; T. S. Azarashvili (555-565).
The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca2+ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.
Keywords: mitochondria; myelin; permeability transition pore; myelin basic protein; CNPase

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A, B, and D by Yu. I. Pobolelova; M. M. Ulyashova; M. Yu. Rubtsova; A. M. Egorov (566-570).
Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.
Keywords: multiplex PCR; carbapenemases; hybridization analysis; DNA microarray

In liver mitochondria loaded with Ca2+ or Sr2+, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca2+-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca2+ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca2+ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca2+ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca2+ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca2+ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca2+ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca2+ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.
Keywords: liver mitochondria; α,ω-hexadecanedioic acid; cyclosporin A-insensitive permeability; Ca2+ ; cytochrome c

There are two physical processes that influence the spatial distribution of transcription factor molecules entering the nucleus of a eukaryotic cell, the binding to genomic DNA and the diffusion throughout the nuclear volume. Comparison of the DNA-protein association rate constant and the protein diffusion constant may determine which one is the limiting factor. If the process is diffusion-limited, transcription factor molecules are captured by DNA before their even distribution in the nuclear volume. Otherwise, if the reaction rate is limiting, these molecules diffuse evenly and then find their binding sites. Using well-studied human NF-κB dimer as an example, we calculated its diffusion constant using the Debye-Smoluchowski equation. The value of diffusion constant was about 10−15 cm3/s, and it was comparable to the NF-κB association rate constant for DNA binding known from previous studies. Thus, both diffusion and DNA binding play an equally important role in NF-κB spatial distribution. The importance of genome 3D-structure in gene expression regulation and possible dependence of gene expression on the local concentration of open chromatin can be hypothesized from our theoretical estimate.
Keywords: transcription factor; nuclear factor κB; diffusion; rate constant; DNA-protein interaction