Biochemistry (Moscow) (v.73, #11)

Role of acidosis, NMDA receptors, and acid-sensitive ion channel 1a (ASIC1a) in neuronal death induced by ischemia by N. K. Isaev; E. V. Stelmashook; E. Y. Plotnikov; T. G. Khryapenkova; E. R. Lozier; Y. V. Doludin; D. N. Silachev; D. B. Zorov (1171-1175).
This review collects data on the influence of intracellular and extracellular acidosis on neuronal viability and the effect of acidosis on neuronal damage progressing under brain ischemia/hypoxia. Particular attention is devoted to the involvement of ionotropic glutamic receptors and acid-sensitive ion channel 1a in these processes.
Keywords: acidosis; neurons; ischemia/hypoxia; pH; ASIC1a

Changes in physicochemical parameters and α-crystallin expression in the lens during cataract development in OXYS rats by Yu. V. Rumyantseva; A. Zh. Fursova; L. A. Fedoseeva; N. G. Kolosova (1176-1182).
The pathogenesis of cataract is associated with oxidative stress and with altered crystallin expression but it is still understood incompletely. In this study, the senescence-accelerated OXYS rats were used as a model. The first biomicro-scopic signs of cataract in OXYS rats were registered at the age of 1.5 months; at 3 months morbidity reached 90%, and at 6 months it reached 100%. Cataract manifestation progresses: at 24 months mature cataract was detected in 90% of eyes of OXYS rats, whereas in 80% of Wistar rat eyes only initial signs of this disease were detected. Analysis of lens redox-parameters has shown that in OXYS rats the intensity of tryptophan fluorescence is higher, the GSH content being higher at 2 months but during formation of mature cataract at 13, 18, and 24 months being lower than in Wistar rats. Decrease in solubility of OXYS rat lens proteins was observed at the age of 13 months. At the age of 3 months gene expression of αA-crystallin and αB-crystallin was 3-fold and 25% lower, respectively, than in Wistar rats. At the age of 14 months there was a 27-fold decrease in expression of αB-crystallin in OXYS rats and it became 21-fold lower than in control. Proteins are synthesized in lens epithelial cells and dystrophic changes in senile cataract result in decrease in structural protein expression. The changes observed in OXYS rats are evidently associated with the dystrophic changes in lens epithelium, which we have described earlier, and are consistent with the model of senile cataract.
Keywords: cataract; oxidative stress; α-crystallin expression; senescence-accelerated OXYS rats

Functional significance of a putative Sp1 transcription factor binding site in the survivin gene promoter by M. V. Mityaev; E. P. Kopantzev; A. A. Buzdin; T. V. Vinogradova; E. D. Sverdlov (1183-1191).
We sequenced 1500-bp genomic DNA regions upstream from the survivin gene (BIRC5). DNA was isolated from human placenta and tumors of patients with diagnosed squamous cancer of the lung that showed high-level BIRC5 gene expression. We have revealed four new promoter allelic variants differing in single nucleotide substitutions, one variant with two nucleotide substitutions, and a variant with a TAAA tetranucleotide insertion. All promoter variants displayed low activity in cells with functionally active p53 protein and high activity in cell lines characterized by low level or absence of p53 protein function. The activity of the promoters with single nucleotide substitutions was comparable to that of the wild-type promoter, whereas two nucleotide substitutions markedly reduced the activity. We also demonstrated the functional significance of a putative Sp1 transcription factor-binding site at (−63...−54) upstream from the transcription initiation site. Mutation within this sequence led to a sharp decrease of promoter activity. The functional architecture of the survivin promoter is discussed based on results known from the literature and those obtained here.
Keywords: survivin promoter; regulation of promoter activity; Sp1 binding sites; SNP

This work deals with 3C (Chromosome Conformation Capture) analysis of the chicken α-globin gene domain in embryonic erythrocytes and lymphoid cells. Ligation products were quantitatively analyzed by real-time PCR with TaqMan probes. It was found that in lymphoid cells, where α-globin gene is not active, the domain has a relatively extended configuration. In embryonic erythrocytes that transcribe αD and αA genes, simultaneous interaction of several domain elements was revealed including the major regulatory element, the erythroid-specific DNase I hypersensitive site at a distance of 9 kb upstream from the α-globin gene cluster (-9 DHS), promoter of the housekeeping gene CGTHBA, the αD-globin gene promoter, and the erythroid-specific enhancer located after the α-globin gene cluster. We suppose that such interaction is necessary to provide for the active transcription status of the chicken α-globin gene domains in erythroid cells.
Keywords: chicken α-globin gene domain; genome spatial organization; 3C; real-time PCR with TaqMan probes

Comparative plasma membrane-associated proteomics of immortalized human hepatocytes by Lan-Tu Gou; Ai-Ping Tong; Li-Juan Chen; Ming-Hai Tang; Bin Chen; Shu-Fang Liang; Canhua Huang; Yu-Quan Wei (1200-1206).
This work was initiated with the purpose of purifying and identifying differentially expressed plasma membrane-associated proteins between human liver cancer cell line HepG2 and normal liver cell line L02. The combined strategy of sucrose density gradient centrifugation and subsequent phase partition was applied to obtain high-purity proteins of plasma membrane. Two-dimensional gel electrophoresis revealed the differential protein profile between the two cell lines. A total of 13 plasma membrane-associated proteins containing 10 up-regulated proteins and three down-regulated proteins in HepG2 cells were successfully identified by MALDI-Q-TOF mass spectrometry; they participate in multiple biological functions such as adhesion, proliferation, apoptosis, and signal transduction. The identified proteins could provide helpful reference in clinical investigations on potential candidates for diagnosis and therapy of liver cancer.
Keywords: plasma membrane; liver cancer; sucrose density gradient centrifugation; two-dimensional gel electrophoresis; proteomics; MALDI-Q-TOF

DNA polymerases β and λ as potential participants of TLS during genomic DNA replication on the lagging strand by A. A. Shtygasheva; E. A. Belousova; N. I. Rechkunova; N. A. Lebedeva; O. I. Lavrik (1207-1213).
The main strategy used by pro-and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases β and λ on DNA substrates using mono-or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg2+ or Mn2+. DNA polymerases β and λ were able to catalyze DNA synthesis across THF only in the presence of Mn2+. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase β. As for DNA polymerase λ, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase β. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase λ. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases β and λ on DNA templates containing damage.
Keywords: DNA replication on lagging strand; translesion synthesis (TLS); AP-sites; DNA gaps; DNA polymerases β and λ; replication protein A (RPA)

Cloning and functional identification of two novel BRCA1 splicing variants by Lixia Miao; Zhijian Cao; Chao Shen; Meijia Gu; Wanhong Liu; Hua Li; Congyi Zheng (1214-1223).
BRCA1 is an important tumor suppressor gene associated with inherited breast and ovarian cancers. In this investigation, two novel BRCA1 splicing variants were cloned from breast cancer cell line ZR-75-30. These transcripts, named BRCA1-PI21-Δ2-21 and BRCA1-Δ2-14, lacked most exons of full length BRCA1 gene, but maintained the original reading frame. We also demonstrated the presence of BRCA1-PI21-Δ2-21 in several human cell lines. Expression of both variants fused with green fluorescent protein (GFP) showed that they targeted different subcellular compartments in the transfected cells. Viability of the cells expressing both fusion proteins decreased notably compared with the viability of cells expressing only GFP. Fluorescence activated cell sorting assay confirmed that the overexpression of two splicing variants resulted in cell apoptosis. Taken together, the different subcellular localization and cell effects of two BRCA1 splicing variants imply that they can have different biological functions in breast cancer cells. Elucidating the functions of BRCA1 splicing variants would help to understand the exact roles of the BRCA1 gene in tumor suppression.
Keywords: breast cancer; BRCA1; alternative splicing; subcellular localization; apoptosis

Efflux of potassium ions from cells and spheroplasts of Saccharomyces cerevisiae yeast treated with silver and copper ions by V. M. Vagabov; A. Yu. Ivanov; T. V. Kulakovskaya; E. V. Kulakovskaya; V. V. Petrov; I. S. Kulaev (1224-1227).
Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H+-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K+ transport systems during preparation of spheroplasts.
Keywords: potassium; silver; copper; cell wall; plasma membrane; H+-ATPase; ion leakage; yeast Saccharomyces cerevisiae

In the absence of exogenous polyamines, the polyamine-deficient Escherichia coli mutant shows not only a characteristic dual-phase growth with abnormal growth, growth arrest, and normal growth after mutation, but also a higher expression of the SOS genes than the polyamine-proficient wild type. The interval of the growth arrest is inversely regulated in a polyamine concentration-dependent manner. These results indicate that the polyamines can act as a signal not only for provoking an adaptive mutation, but also for hastening generation of an adaptive mutation.
Keywords: adaptive mutation; Escherichia coli ; mutational speed; polyamine; SOS response

Incubation of Aspergillus niger glucose oxidase with glucose, fructose, or ribose results in remarkable inactivation of the enzyme. Glucose oxidase incubated with the sugars migrated as a diffuse band of low intensity and silver stained poorly after SDS-PAGE. Purified anti-glucose oxidase antibodies and F(ab)′2 or F(ab)′ derived therefrom were effective in restricting the inactivation of the enzyme induced by the sugars, providing up to 90% protection. The sugars also caused remarkable changes in the electrophoretic behavior of anti-glucose oxidase antibodies and the fragments, but complexing with glucose oxidase restricted the changes both in the enzyme and the antibody/antibody fragments.
Keywords: antigen-antibody complex; anti-glycation agents; F(ab)′2 ; F(ab)′; glycation

Antibodies against DNA hydrolyze DNA and RNA by M. A. Krasnorutskii; V. N. Buneva; G. A. Nevinsky (1242-1253).
In this work, rabbits were immunized with a high polymer DNA complexed with methylated BSA (mBSA) and by mBSA. It is shown that electrophoretically homogeneous preparations of polyclonal antibodies (Ab) from non-immunized rabbits and animals immunized by mBSA do not exhibit catalytic activity. Ab from the blood of rabbits immunized with the DNA-mBSA complex hydrolyzed poly(C) and different RNAs with efficiency exceeding that towards DNA by approximately 3–4 orders of magnitude. Affinity chromatography of the IgG on DNA cellulose separated the Ab into fractions hydrolyzing both RNA and DNA, and for the first time fractions that hydrolyze only RNA were found. Kinetic parameters that characterize the RNA and DNA hydrolysis by initial Ab preparations and their fractions obtained by separation on an affinity sorbent are compared.
Keywords: rabbit immunization by DNA; DNA-and RNA-hydrolyzing abzymes

Mitochondrial matrix fragmentation as a protection mechanism of yeast Saccharomyces cerevisiae by D. A. Knorre; S. M. Ojovan; V. B. Saprunova; S. S. Sokolov; L. E. Bakeeva; F. F. Severin (1254-1259).
It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non-fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Δysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.
Keywords: yeast; mitochondrion; mitochondria fragmentation; ysp2 ; amiodarone; apoptosis

Identification of recognition sites for Myc/Max/Mxd network proteins by a whole human chromosome 19 selection strategy by S. B. Akopov; I. P. Chernov; T. Wahlström; M. B. Kostina; G. Klein; M. Henriksson; L. G. Nikolaev (1260-1268).
In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up-or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors.
Keywords: Max; Myc; Mxd; target genes; human chromosome 19

Human biochemistry and disease by Gherman Wiederschain (1269-1270).