Mitochondrial Dysfunction Due to Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis

doi:10.1089/ars.2015.6318

. 2015 Nov 10;23(14):1059-75.

doi: 10.1089/ars.2015.6318. Epub 2015 Nov 5.

Mitochondrial Dysfunction Due to Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis

Anja Konzack¹, Mirza Jakupovic², Kateryna Kubaichuk¹, Agnes Görlach³, Frank Dombrowski⁴, Ilkka Miinalainen⁵, Raija Sormunen⁵, Thomas Kietzmann¹

Affiliations

¹ 1 Faculty of Biochemistry and Molecular Medicine, and Biocenter Oulu, University of Oulu , Oulu, Finland .
² 2 Department of Chemistry, University of Kaiserslautern , Kaiserslautern, Germany .
³ 3 Experimental and Molecular Pediatric Cardiology, German Heart Center Munich, Technical University Munich , Munich, Germany .
⁴ 4 Institute of Pathology, University of Greifswald , Greifswald, Germany .
⁵ 5 Biocenter Oulu Electron Microscopy Core Facility, University of Oulu , Oulu, Finland .

PMID: 26422659
PMCID: PMC4657515
DOI: 10.1089/ars.2015.6318

Mitochondrial Dysfunction Due to Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis

Anja Konzack et al. Antioxid Redox Signal. 2015.

. 2015 Nov 10;23(14):1059-75.

doi: 10.1089/ars.2015.6318. Epub 2015 Nov 5.

Authors

Anja Konzack¹, Mirza Jakupovic², Kateryna Kubaichuk¹, Agnes Görlach³, Frank Dombrowski⁴, Ilkka Miinalainen⁵, Raija Sormunen⁵, Thomas Kietzmann¹

Affiliations

¹ 1 Faculty of Biochemistry and Molecular Medicine, and Biocenter Oulu, University of Oulu , Oulu, Finland .
² 2 Department of Chemistry, University of Kaiserslautern , Kaiserslautern, Germany .
³ 3 Experimental and Molecular Pediatric Cardiology, German Heart Center Munich, Technical University Munich , Munich, Germany .
⁴ 4 Institute of Pathology, University of Greifswald , Greifswald, Germany .
⁵ 5 Biocenter Oulu Electron Microscopy Core Facility, University of Oulu , Oulu, Finland .

PMID: 26422659
PMCID: PMC4657515
DOI: 10.1089/ars.2015.6318

Abstract

Aims: One of the cancer hallmarks is mitochondrial dysfunction associated with oxidative stress. Among the first line of defense against oxidative stress is the dismutation of superoxide radicals, which in the mitochondria is carried out by manganese superoxide dismutase (MnSOD). Accordingly, carcinogenesis would be associated with a dysregulation in MnSOD expression. However, the association studies available so far are conflicting, and no direct proof concerning the role of MnSOD as a tumor promoter or suppressor has been provided. Therefore, we investigated the role of MnSOD in carcinogenesis by studying the effect of MnSOD deficiency in cells and in the livers of mice.

Results: We found that loss of MnSOD in hepatoma cells contributed to their conversion toward a more malignant phenotype, affecting all cellular properties generally associated with metabolic transformation and tumorigenesis. In vivo, hepatocyte-specific MnSOD-deficient mice showed changed organ architecture, increased expression of tumor markers, and a faster response to carcinogenesis. Moreover, deficiency of MnSOD in both the in vitro and in vivo model reduced β-catenin and hypoxia-inducible factor-1α levels.

Innovation: The present study shows for the first time the important correlation between MnSOD presence and the regulation of two major pathways involved in carcinogenesis, the Wnt/β-catenin and hypoxia signaling pathway.

Conclusion: Our study points toward a tumor suppressive role of MnSOD in liver, where the Wnt/β-catenin and hypoxia pathway may be crucial elements.

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Figures

<b>FIG. 1.</b> — **FIG. 1.**
**Knockdown of MnSOD induces ROS levels. (A)** HepG2 cells were stably transfected with a scrambled (sc) shRNA or shRNA against MnSOD (kd). Protein lysates were prepared and analyzed by Western blotting with antibodies against MnSOD, Sod-1, Catalase, GPx-1, Prdx1, Prdx3, Trn2, Nrf-2, HO-1, and α-tubulin. Data are mean ± SD of fold induction normalized to HepG2-sc cells (n = 3, *p < 0.05) **(B)** Representative Western blots. α-tubulin is shown for equal loading of proteins. (C–E) The redox state of HepG2-sc and MnSOD-kd cells was determined by measuring **(C)** DHE fluorescence (n = 3), **(D)** DHE fluorescence microscopy showing mitochondrial localization of superoxide (n = 3), **(E)** DCF fluorescence (n = 10), and **(F)** GSH levels (n = 3) (c.f. section “Materials and Methods”). Data are mean ± SD of fold induction normalized to HepG2-sc (*p < 0.05). **(G)** Nrf2 localization analyzed by confocal fluorescence immunohistochemistry. **(H)** Representative Western blot showing Nrf2 localization after cell fractionation; Lamin A/C is shown to indicate nuclear fractions, and α-tubulin is shown for cytosolic fraction. DCF, dichlorodihydrofluorescein; DHE, dihydroethidium; Gpx-1, glutathione peroxidase-1; GSH, reduced glutathionel; HO-1, heme oxygenase-1; kd, knockdown; MnSOD, manganese superoxide dismutase; Nrf2, NF-E2-related factor 2; Prdx, peroxiredoxin; ROS, reactive oxygen species; SD, standard deviation. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 2.</b> — **FIG. 2.**
**MnSOD knockdown alters mitochondrial function. (A)** Transmission electron microscopy images of HepG2-sc and MnSOD-kd cells at 1200× and 4800× magnification. **(B)** Comparison of intracellular mitochondrial networks in HepG2-sc and MnSOD-kd cells visualized by TMRE (200 nM) staining and fluorescence microscopy. **(C)** Mitochondrial membrane potential (ΔΨ_m) was assessed by TMRE fluorescence using DNP as an uncoupler. Results are shown as percentage normalized to the HepG2-sc control and presented as mean ± SD; *Significant difference sc *versus* kd, p < 0.05. **(D)** Oxygen consumption of HepG2-sc and MnSOD-kd cells was measured by high-resolution respirometry. Measured were routine (R) respiration, proton leak (L) respiration after oligomycin-initiated inhibition of ATP synthase (5 mM oligomycin), ETC capacity (E) at FCCP-uncoupled respiration (5–7 mM FCCP), and nonmitochondrial respiration (rox) after inhibition of respiratory chain complexes I and III by rotenone (1 mM) and antimycin A (5 mM). Data were normalized to cell number, and the relative changes are presented. Abbreviations: R/E = ratio of routine and noncoupled respiration, L/E = ratio of oligomycin-inhibited and noncoupled respiration, rox/E′ = rate of oxidative phosphorylation-independent oxygen consumption (*Significant difference p < 0.05). DNP, 2,4-dinitrophenol; ETC, electron transport chain; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; TMRE, tetramethylrhodamine, ethyl ester. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 3.</b> — **FIG. 3.**
**Loss of MnSOD alters cell morphology and increases proliferation in HepG2 cells. (A)** Representative images of HepG2-sc (sc) and MnSOD-kd (kd) cells stained with crystal violet and DAPI. Images were taken using a 60×/1.00 oil immersion objective in bright-field to visualize crystal violet staining and fluorescence at an appropriate filter set for DAPI. **(B)** The effect of MnSOD deficiency on the proliferative ability was measured by counting of cells, BrdU assay, and MTT assay. Data are presented as fold induction (mean ± SD) normalized to HepG2-sc cells (*p < 0.05). **(C)** FACS analysis of apoptotic and necrotic cells after double staining with Annexin-V-FLOUS (AV) and PI at a sampling rate of 1 ml/min and wavelengths of λ_ex = 488 nm, λ_em = 518 nm for AV and λ_ex = 488 nm, λ_em = 617 nm for PI. Representative plot displaying AV *versus* PI shows three clearly separated populations of cells: R2 = live (AV neg, PI neg), R3 = early apoptotic (AV pos, PI neg), and R4 = late apoptotic/necrotic (AV pos, PI pos). **(D)** Alive (h), early apoptotic (ea), and late apoptotic/necrotic (a/n) cells analyzed by FACS. Data are mean ± SD of the fold induction normalized to HepG2-sc cells. BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 4.</b> — **FIG. 4.**
**MnSOD depletion affects anchorage-independent and anchorage-dependent cell growth. (A)** Anchorage-independent and anchorage-dependent growth of HepG2-sc and MnSOD-kd cells was assessed by colony formation and soft agar assay, respectively. Data are presented as sum of colony volume [Σ(V)] and sum of area [Σ(A)], respectively. **(B)** Representative images from soft agar and colony formation assay are shown. **(C)** Migration was determined by a transwell assay (8 μm pore size) under serum starvation. Data are mean ± SD of the fold induction normalized to HepG2-sc cells. Representative images and quantification of migrated cells are shown. **(D)** Cell adhesion was assessed by rotating cells at 250 rpm on an orbital shaker for 2 h at 37°C, while keeping a control plate motionless. The number of adherent cells in the motionless control plates was set to 100%; data are mean ± SD (*Significant difference, p < 0.05). **(E)** Representative images of crystal violet stained cells still attached to the plate are shown. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 5.</b> — **FIG. 5.**
**Hepatocyte-specific MnSOD KO in mice induces 8-isoprostane. (A)** Immunoblot analyses for MnSOD in protein lysates prepared from liver, kidney, and brain of the MnSOD^flox/flox control mice (WT) or hepatocyte-specific MnSOD-KO mice. One hundred microgram of protein was subjected to Western blot analyses with antibodies against MnSOD and β-actin. The β-actin is shown for equal loading of proteins. **(B)** Scheme of the superoxide-mediated peroxidation of an arachidonic acid (Ara) containing lipid. **(C)** Liver sections from 3-month-old, male MnSOD^flox/flox control mice (WT) and MnSOD-KO mice (KO) were prepared and immunostained for 8-isoprostane. 400× magnification. **(D)** 8-Isoprostane staining normalized to WT and presented as mean ± SD (n = 6/group; *p < 0.05). KO, knockout; WT, wild type. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 6.</b> — **FIG. 6.**
**Hepatocyte-specific MnSOD-KO in mice induces 3-nitrotyrosine formation. (A)** Scheme of the superoxide-mediated formation of nitrotyrosine. **(B)** Liver sections from 3-month-old, male MnSOD^flox/flox control mice (WT) and MnSOD-KO mice (KO) were prepared and immunostained for 3-nitrotyrosine. 40× magnification. **(C)** 3-Nitrotyrosine formation normalized to WT and presented as mean ± SD (n = 6/group; *p < 0.05). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 7.</b> — **FIG. 7.**
**Lack of MnSOD in hepatocytes causes liver damage. (A)** Livers from 3-month-old, male MnSOD^flox/flox control mice (WT) and MnSOD-KO mice (KO) were prepared and analyzed for morphological changes. Cells showing mitotic signs, inflammatory infiltrates, and lipid droplets were counted using a ×400 magnification (0.15 mm² field), and the respective index (nuclei with visible chromosomes/total number of nuclei with observations at least 5000 counted nuclei) as well as the Knodell fibrosis score (33) compared to the WT animals, was determined. Data presented as mean ± SD (n = 6/group; *p < 0.05). **(B)** Representative liver sections from wild-type MnSOD^flox/flox (WT) and hepatocyte-specific MnSOD-KO mice stained with hematoxylin/eosin. The livers of hepatocyte-specific MnSOD-KO mice displayed sings of inflammation (inflammatory foci in the *middle panel* in KO). In addition, an enhanced number of mitotic cells (*arrow* in *left* KO) and lipid droplet containing cells (*right* KO) were present in these animals. **(C)** Blood was drawn from 3-month-old, male control MnSOD^flox/flox mice (WT) and MnSOD-KO mice (KO); the individual samples of serum were analyzed for different parameters indicating liver injury. The respective levels or activities in the WT animals were set to 1 (*Significant difference p < 0.05). **(D)** Serum parameters in the WT and KO mice, values represent mean ± SD (n = 6/group; *p < 0.05,). Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase (formerly GPT, glutamate pyruvate aminotransferase); AST, aspartate aminotransferase (formerly GOT, glutamate oxaloacetate aminotransferase); Bili, bilirubin; Che, choline esterase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 8.</b> — **FIG. 8.**
**Loss of MnSOD in hepatocytes promotes tumorigenesis. (A)** Livers from 3-month-old, male MnSOD^flox/flox control mice (WT) and MnSOD-KO mice (KO) were prepared and immunostained for GST-P and GS, and positive cells were counted using a 40× magnification. Data represented as positive cells per 100 hepatocytic nuclei; at least 5000 nuclei have been counted (n = 6/group; *p < 0.05). **(B)** Representative liver sections from wild-type MnSOD^flox/flox (WT) and hepatocyte-specific MnSOD-KO mice immunostained with antibodies against GST-P or GS as early tumor markers. *Dark brown* precipitates indicate the GST- or GS-positive cells. 40× magnification. **(C)** Diethylnitrosamine induced liver tumors in mice. **(i)** Massive DEN-induced liver tumorigenesis in hepatocyte-specific MnSOD-KO mice 12 months after DEN injection (Table 1). **(ii)** Large areas of perivenular chronic inflammation in the livers of KO mice compared to normal morphology of WT animals 6 months after DEN treatment; 40×. **(iii)** Preneoplastic lesions in WT and KO mice 9 and 6 months after DEN injection, respectively; 200×. **(iv)** Oval cells in WT compared to liver adenoma in KO liver tissue 12 months after DEN treatment; 400×. Hematoxylin–Eosin staining. DEN, diethylnitrosamine; GS, glutamine synthetase; GST-P, placental glutathione S-transferase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 9.</b> — **FIG. 9.**
**Lack of MnSOD decreases APC, β-catenin, and HIF-1α levels** ***in vivo*** **in mice and** ***in vitro*** **in HepG2 cells. (A)** Representative liver sections from 3-month-old, male wild-type MnSOD^flox/flox (WT) and hepatocyte-specific MnSOD-KO mice immunostained for APC, β-catenin, and HIF-1α. *Dark* areas indicate high APC, β-catenin, and HIF-1α expression. **(B)** Quantification of immunostained tissue sections of 3-month-old animals (n = 4/group; *p < 0.05). **(C)** HepG2 cells stably transfected with a scrambled (sc) shRNA or shRNA against MnSOD (kd) were exposed to normoxia 16% O₂ or hypoxia 5% O₂ for 4 h. Thereafter, protein lysates were prepared and analyzed by Western blotting with antibodies against APC, β-catenin, HIF-1α, and α-tubulin, respectively. α-Tubulin is shown for equal loading of proteins. *Arrows* indicate areas of changed expression. APC, adenoma polyposis coli; HIF-1α, hypoxia-inducible transcription factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

<b>FIG. 10.</b> — **FIG. 10.**
**MnTBAP rescues β-catenin and HIF-1α activity in MnSOD lacking cells, whereas knockdown of β-catenin and HIF-1α mimics lack of MnSOD in HepG2-sc cells. (A)** HepG2-sc or MnSOD-kd cells were transiently transfected with either TopFlash or HRE-Luc gene constructs and were cotransfected with shRNAs against β-catenin and HIF-1α, or treated with MnTBAP (100 μM) or NAC (1 mM) for 24 h. Luc activity levels were normalized to Renilla luciferase levels. The percentage of Luc activity was determined relatively to corresponding controls that were set equal to 100%. Data are mean ± SD (*Significant difference, p < 0.05). **(B)** MnTBAP rescues the GSH levels in MnSOD-kd cells. HepG2-sc and MnSOD-kd cells were treated with MnTBAP (100 μM) for 24 h, and GSH levels were measured. Data are mean ± SD (*Significant difference, p < 0.05). **(C)** Knockdown of β-catenin and HIF-1α mimics the proliferative phenotype of MnSOD lacking cells. HepG2-sc cells were transfected with shRNAs against HIF-1α or β-catenin, controls were cotransfected with an empty vector, and BrdU assays were performed. Data are presented as fold induction (mean ± SD) normalized to HepG2-sc cells, where the BrdU level was set to 100% (*Significant difference, p < 0.05). NAC, N-acetylcysteine; MnTBAP, Mn(III) tetrakis (4-benzoic acid) porphyrin chloride.

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References

1. Aniya Y. and Anders MW. Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species. J Biol Chem 264: 1998–2002, 1989 - PubMed
1. Borgstahl GEO, Parge HE, Hickey MJ, Johnson MJ, Boissinot M, Hallewell RA, Lepock JR, Cabelli DE, and Tainer JA. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry (N Y) 35: 4287–4297, 1996 - PubMed
1. Bravard A, Sabatier L, Hoffschir F, Ricoul M, Luccioni C, and Dutrillaux B. SOD2: a new type of tumor-suppressor gene? Int J Cancer 51: 476–480, 1992 - PubMed
1. Burke ZD, Reed KR, Phesse TJ, Sansom OJ, Clarke AR, and Tosh D. Liver zonation occurs through a β-catenin-dependent, c-Myc-INDEPENDENT mechanism. Gastroenterology 136: 2316–2324.e3, 2009 - PubMed
1. Burke ZD. and Tosh D. The Wnt/β-catenin pathway: master regulator of liver zonation? Bioessays 28: 1072–1077, 2006 - PubMed

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[1] Aniya Y. and Anders MW. Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species. J Biol Chem 264: 1998–2002, 1989 - PubMed

[2] Aniya Y. and Anders MW. Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species. J Biol Chem 264: 1998–2002, 1989 - PubMed

[3] Borgstahl GEO, Parge HE, Hickey MJ, Johnson MJ, Boissinot M, Hallewell RA, Lepock JR, Cabelli DE, and Tainer JA. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry (N Y) 35: 4287–4297, 1996 - PubMed

[4] Borgstahl GEO, Parge HE, Hickey MJ, Johnson MJ, Boissinot M, Hallewell RA, Lepock JR, Cabelli DE, and Tainer JA. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry (N Y) 35: 4287–4297, 1996 - PubMed

[5] Bravard A, Sabatier L, Hoffschir F, Ricoul M, Luccioni C, and Dutrillaux B. SOD2: a new type of tumor-suppressor gene? Int J Cancer 51: 476–480, 1992 - PubMed

[6] Bravard A, Sabatier L, Hoffschir F, Ricoul M, Luccioni C, and Dutrillaux B. SOD2: a new type of tumor-suppressor gene? Int J Cancer 51: 476–480, 1992 - PubMed

[7] Burke ZD, Reed KR, Phesse TJ, Sansom OJ, Clarke AR, and Tosh D. Liver zonation occurs through a β-catenin-dependent, c-Myc-INDEPENDENT mechanism. Gastroenterology 136: 2316–2324.e3, 2009 - PubMed

[8] Burke ZD, Reed KR, Phesse TJ, Sansom OJ, Clarke AR, and Tosh D. Liver zonation occurs through a β-catenin-dependent, c-Myc-INDEPENDENT mechanism. Gastroenterology 136: 2316–2324.e3, 2009 - PubMed

[9] Burke ZD. and Tosh D. The Wnt/β-catenin pathway: master regulator of liver zonation? Bioessays 28: 1072–1077, 2006 - PubMed

[10] Burke ZD. and Tosh D. The Wnt/β-catenin pathway: master regulator of liver zonation? Bioessays 28: 1072–1077, 2006 - PubMed

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Mitochondrial Dysfunction Due to Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis

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Mitochondrial Dysfunction Due to Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis

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