Biochemistry (Moscow) (v.81, #6)
Molecular and cellular bases of iron metabolism in humans by I. V. Milto; I. V. Suhodolo; V. D. Prokopieva; T. K. Klimenteva (549-564).
Iron is a microelement with the most completely studied biological functions. Its wide dissemination in nature and involvement in key metabolic pathways determine the great importance of this metal for uniand multicellular organisms. The biological role of iron is characterized by its indispensability in cell respiration and various biochemical processes providing normal functioning of cells and organs of the human body. Iron also plays an important role in the generation of free radicals, which under different conditions can be useful or damaging to biomolecules and cells. In the literature, there are many reviews devoted to iron metabolism and its regulation in proand eukaryotes. Significant progress has been achieved recently in understanding molecular bases of iron metabolism. The purpose of this review is to systematize available data on mechanisms of iron assimilation, distribution, and elimination from the human body, as well as on its biological importance and on the major iron-containing proteins. The review summarizes recent ideas about iron metabolism. Special attention is paid to mechanisms of iron absorption in the small intestine and to interrelationships of cellular and extracellular pools of this metal in the human body.
Keywords: oxidized/reduced; intracellular/extracellular; heme/nonheme; exogenous/endogenous iron
Type 1 metallothionein (ZjMT) is responsible for heavy metal tolerance in Ziziphus jujuba by Lan-Song Li; Yu-Ping Meng; Qiu-Fen Cao; Yong-Zhen Yang; Fan Wang; Hu-Sheng Jia; Shu-Biao Wu; Xu-Guang Liu (565-573).
Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that are able to make cells to uptake heavy metals from the environment. Molecular and functional characterization of this gene family improves understanding of the mechanisms underlying heavy metal tolerance in higher organisms. In this study, a cDNA clone, encoding 74-a.a. metallothionein type 1 protein (ZjMT), was isolated from the cDNA library of Ziziphus jujuba. At the N- and C-terminals of the deduced amino acid sequence of ZjMT, six cysteine residues were arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC structure, respectively, indicating that ZjMT is a type 1 MT. Quantitative PCR analysis of plants subjected to cadmium stress showed enhanced expression of ZjMT gene in Z. jujuba within 24 h upon Cd exposure. Escherichia coli cells expressing ZjMT exhibited enhanced metal tolerance and higher accumulation of metal ions compared with control cells. The results indicate that ZjMT contributes to the detoxification of metal ions and provides marked tolerance against metal stresses. Therefore, ZjMT may be a potential candidate for tolerance enhancement in vulnerable plants to heavy metal stress and E. coli cells containing the ZjMT gene may be applied to adsorb heavy metals in polluted wastewater.
Keywords: Z. jujuba Mill.; metallothionein; heavy metal tolerance; heavy metal hyperaccumulation
Upregulation of p72 enhances malignant migration and invasion of glioma cells by repressing Beclin1 expression by Zhenxing Zhang; He Tian; Ye Miao; Xu Feng; Yang Li; Honglei Wang; Xiaofeng Song (574-582).
p72 is the member of the DEAD-box RNA helicase family, which can unwind double-stranded RNA and is efficient for microRNA (miRNA, miR) processing. However, its specific role in glioma has not been elucidated. First, the expression of p72 in glioma cell lines and tissues was explored using Western blot. To explore the role of p72 on glioma progression, adenovirus inhibiting p72 was transfected into A172 and T98G cells. Cell autophagy was determined using GFPLC3 dots, and cell apoptosis was determined using flow cytometry. The effect of Beclin1 was explored using GFP-LC3 dots, flow cytometry, and colony formation. The possible miRNAs that target the 3′-untranslated region (3′-UTR) of Beclin1 were predicted using TargetScan. Dual luciferase reporter assay was applied to determine whether these miRNAs bind to the 3′-UTR of Beclin1. The expression of p72 was significantly increased in glioma cell lines and tissues. Autophagy-related protein Beclin1 was found to be significantly enhanced when p72 was inhibited. The accumulation of GFP-LC3 dots was significant in cells transfected with ad-sh-p72 compared with ad-con. Colony formation capacity and cell apoptosis were also found to be significantly decreased with p72 inhibition. Furthermore, upregulation of Beclin1 contributes to A172 cell autophagy, invasion, and apoptosis. Overexpression of p72 induces increased miR-34-5p and miR-5195-3p expression in A172 and T98G cells. Beclin1 was the target gene of miR-34-5p and miR-5195-3p. In conclusion, we found for the first time that overexpression of p72 decreased Beclin1 expression partially by increasing miR-34-5p and miR-5195-3p expression in A172 and T98G cells.
Keywords: glioma; p72; miR-34-5p; miR-5195-3p; Beclin1
Non-photochemical fluorescence quenching in photosystem II antenna complexes by the reaction center cation radical by V. Z. Paschenko; V. V. Gorokhov; N. P. Grishanova; B. N. Korvatovskii; M. V. Ivanov; E. G. Maksimov; M. D. Mamedov (583-590).
In direct experiments, rate constants of photochemical (k P) and non-photochemical (k P + ) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the “dark” reaction center (RC) state, antenna fluorescence decay kinetics were measured under lowintensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a “closed” [P680+Q A – ] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680+Q A – ] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680+) state was found to be ∼1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ1 obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680+Pheo– charge recombination. Thus, for the first time the ratio of k P + /k P = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.
Keywords: photosystem II; non-photochemical quenching; photochemical reactions; light-harvesting antenna; reaction center
CELSR1 is a positive regulator of endothelial cell migration and angiogenesis by Yi-Hong Zhan; Qi-Cong Luo; Xiao-Rong Zhang; Nai-An Xiao; Cong-Xia Lu; Cen Yue; Ning Wang; Qi-Lin Ma (591-599).
Cadherin is an epidermal growth factor and laminin-G seven-pass G-type receptor 1 (CELSR1) is a key component of the noncanonical Wnt/planar cell polarity (PCP) pathway that critically regulates endothelial cell proliferation and angiogenesis. In this study, we examined the biological significance of CELSR1 in endothelial cell migration and angiogenesis. For this, we applied both gain-of-function and loss-of-function approaches. To increase the endogenous expression of CELSR1, we used the transcription activator-like effector (TALE) technology and constructed an artificial TALE-VP64 activator. To knock down the expression of CELSR1, we generated lentivirus containing short hairpin RNA sequences targeting different regions of CELSR1 mRNA. Following up- or down-regulation of CELSR1 in human aortic endothelial cells (HAEC), we assessed in vitro cell proliferation by MTT assay, migration by scratch and transwell migration assays, and angiogenesis by tube formation analysis. We found that CELSR1 was endogenously expressed in human umbilical vein endothelial cells (HUVEC) and HAEC. When focusing on HAEC, we found that upregulating CELSR1 expression significantly promoted cell growth, while knocking down CELSR1 inhibited the growth (p < 0.05). Using both scratch and transwell migration assays, we observed a positive correlation between CELSR1 expression and cell migratory capability. In addition, CELSR1 upregulation led to higher levels of tube formation in HAEC, while downregulating CELSR1 expression decreased tube formation (p < 0.05). Mechanistically, CELSR1-regulated migration and tube formation was mediated through disheveled segment polarity protein 3 (Dvl3). In conclusion, CELSR1 plays an important role in regulating multiple phenotypes of endothelial cells, including proliferation, migration, and formation of capillary-like structures.
Keywords: CELSR1; endothelial cells; migration; angiogenesis
Structural relationships between genetically closely related O-antigens of Escherichia coli and Shigella spp. by Y. A. Knirel; Chengqian Qian; A. S. Shashkov; O. V. Sizova; E. L. Zdorovenko; O. I. Naumenko; S. N. Senchenkova; A. V. Perepelov; Bin Liu (600-608).
Errors have been found in polysaccharide structures on pages 603 (Fig. 3), 606 (Figs. 13-15) and 607 (Fig. 17). The following are the correct structures.Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using 1H and 13C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.
Keywords: Escherichia coli ; Shigella dysenteriae ; Shigella boydii ; O-polysaccharide structure; O-antigen gene cluster
Priming of human neutrophils is necessary for their activation by extracellular DNA by A. S. Prikhodko; M. V. Vitushkina; L. A. Zinovkina; E. N. Popova; R. A. Zinovkin (609-614).
Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSFinduced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA.
Keywords: neutrophil activation; GM-CSF; extracellular DNA; nuclear DNA; mitochondrial DNA
Mitochondrial dysfunction in neocortex and hippocampus of olfactory bulbectomized mice, a model of Alzheimer’s disease by A. V. Avetisyan; A. N. Samokhin; I. Y. Alexandrova; R. A. Zinovkin; R. A. Simonyan; N. V. Bobkova (615-623).
Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer’s disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aβ) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aβ, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aβ1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals–familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.
Keywords: Alzheimer’s disease; olfactory bulbectomized mice; mitochondrial dysfunction; oxidative stress
Application of created restriction site PCR-RFLP to identify POT1 gene polymorphism by Tuanwei Wang; Sihua Wang; Xiaoran Duan; Xiaolei Feng; Pengpeng Wang; Wu Yao; Yongjun Wu; Feifei Feng; Songcheng Yu; Yiming Wu; Wei Wang (624-627).
Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3′-end of the forward primer was next to the polymorphic site, and the third base from the 3′-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.
Keywords: created restriction site; single nucleotide polymorphism; protection of telomeres 1
Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of YB-1 and LRP/MVP by N. I. Moiseeva; O. Yu. Susova; A. A. Mitrofanov; D. Yu. Panteleev; G. V. Pavlova; N. A. Pustogarov; A. A. Stavrovskaya; E. Yu. Rybalkina (628-635).
Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.
Keywords: glioblastoma; primary cell culture; temozolomide; drug resistance; proliferation; YB-1; MVP
Glycosylphosphatidylinositol-anchored proteins as regulators of cortical cytoskeleton by G. V. Sharonov; M. N. Balatskaya; V. A. Tkachuk (636-650).
Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are important players in reception and signal transduction, cell adhesion, guidance, formation of immune synapses, and endocytosis. At that, a particular GPI-AP can have different activities depending on a ligand. It is known that GPI-AP oligomer creates a lipid raft in its base on plasma membrane, which serves as a signaling platform for binding and activation of src-family kinases. Yet, this does not explain different activities of GPI-APs. Meanwhile, it has been shown that short-lived actomyosin complexes are bound to GPI-APs through lipid rafts. Here, we hypothesize that cell cortical cytoskeleton is the main target of GPI-AP signaling. Our hypothesis is based on the fact that the GPI-AP-induced lipid raft bound to actin filaments and anionic lipids of this raft is known to interact with and activate various actin-nucleating factors, such as formins and N-WASP. It is also known that these and other actin-regulating proteins are activated by src-family kinases directly or through their effectors, such as cortactin and abl-kinases. Regulation of cytoskeleton by GPI-APs may have impact on morphogenesis, cell guidance, and endocytosis, as well as on signaling of other receptors. To evaluate our hypothesis, we have comprehensively considered physiological activities of two GPI-APs–urokinase receptor and T-cadherin.
Keywords: glycosylphosphatidylinositol; cytoskeleton; actin; lipid rafts; uPAR; T-cadherin