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BBA - General Subjects (v.1800, #3)

Editorial Board (pp. i).

Mitochondrial Research and Medicine by Yau-Huei Wei (pp. 203-204).

New extension of the Mitchell Theory for oxidative phosphorylation in mitochondria of living organisms by Bernhard Kadenbach; Rabia Ramzan; Li Wen; Sebastian Vogt (pp. 205-212).

The Mitchell Theory implies the proton motive force Δp across the inner mitochondrial membrane as the energy-rich intermediate of oxidative phosphorylation. Δp is composed mainly of an electrical (ΔΨ_m) and a chemical part (ΔpH) and generated by the respiratory chain complexes I, III and IV. It is consumed mostly by the ATP synthase (complex V) to produce ATP. The free energy of electron transport within the proton pumps is sufficient to generate Δp of about 240 mV. The proton permeability of biological membranes, however, increases exponentially above 130 mV leading to a waste of energy at high values (ΔΨ_m>140 mV). In addition, at ΔΨ_m>140 mV, the production of the superoxide radical anion O₂⁻ at complexes I, II and III increases exponentially with increasing ΔΨ_m. O₂⁻ and its neutral product H₂O₂ (=ROS, reactive oxygen species) induce oxidative stress which participates in aging and in the generation of degenerative diseases. Here we describe a new mechanism which acts independently of the Mitchell Theory and keeps ΔΨ_m at low values through feedback inhibition of complex IV (cytochrome c oxidase) at high ATP/ADP ratios, thus preventing the formation of ROS and maintaining high efficiency of oxidative phosphorylation.

Keywords: Mitochondrial membrane potential; Hyperpolarization; Mitchell Theory; Cytochrome; c; oxidase; Degenerative disease; Oxidative stress; Protein phosphorylation

Evidence for ΔpH surface component (ΔpH^S) of proton motive force in ATP synthesis of mitochondria by Jing-Wei Xiong; Liping Zhu; Xuanmao Jiao; Shu-Sen Liu (pp. 213-222).

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.We measure ∆pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ∆pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ∆pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ∆pH^S, but not with ∆ Ψ_E.Our data substantiate ∆pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ∆pH^S and ∆ Ψ_E in mitochondrial bioenergetics are also presented.

Keywords: Fluorescein-PE labeled mitoplasts; Interfacial proton flow (∆pH; ^S; ); ∆; Ψ; _E; (ΔµH; ⁺; ); ATP synthesis; Mitochondria; Chemiosmotic theory

Transcriptional and post-transcriptional regulation of mitochondrial biogenesis in skeletal muscle: Effects of exercise and aging by Vladimir Ljubicic; Anna-Maria Joseph; Ayesha Saleem; Giulia Uguccioni; Melania Collu-Marchese; Ruanne Y.J. Lai; Linda M.-D. Nguyen; David A. Hood (pp. 223-234).

Acute contractile activity of skeletal muscle initiates the activation of signaling kinases. This promotes the phosphorylation of transcription factors, leading to enhanced DNA binding and transcriptional activation and/or repression. The mRNA products of nuclear genes encoding mitochondrial proteins are translated in the cytosol and imported into pre-existing mitochondria. When contractile activity is repeated, the recapitulation of these cellular events progressively leads to an expansion of the mitochondrial reticulum within muscle. This has physiologically relevant health benefit, including enhanced lipid metabolism and reduced muscle fatigability. In aging skeletal muscle, the response to contractile activity appears to be attenuated, suggesting that a greater contractile stimulus is required to attain a similar phenotype adaptation. This review summarizes our current understanding of the effects of exercise on the gene expression pathway leading to organelle biogenesis in muscle.

Keywords: Abbreviations; Δ; ψ; _m; mitochondrial membrane potential; AAC; ADP/ATP carrier; AIF; apoptosis inducing factor; AKT; protein kinase B; AMPK; AMP-activated protein kinase; ATF2; activating transcription factor 2; AUF1; ARE-associated factor; Ca; ²⁺; calcium; CaMKIV; calcium/calmodulin-dependent protein kinase IV; COX; cytochrome; c; oxidase; CRE; cAMP response element; CREB; CRE binding protein; Egr-1; early growth response gene-1; ERRα; estrogen-related receptor; EDL; extensor digitorum longus; ETC; electron transport chain; FTR; fast-twitch red; FTW; fast-twitch white; GIP; general import pore; HMG; high mobility group; Hsp; heat shock protein; HuR; human-antigen R; IMF; intermyofibrillar; KO; knockout; L-/H-strand; light/heavy-strand; MAPK; mitogen-activated protein kinases; MDH; malate dehydrogenase; MEF2; myocyte enhancer factor 2; MHC; myosin heavy chain; miRNA/miRs; microRNAs; MMD; multiple mitochondrial disease; MSF; mitochondrial import stimulation factor; mtDNA; mitochondrial DNA; NRF-1/2; nuclear respiratory factor-1/2; NUGEMPs; nuclear genes encoding mitochondrial proteins; PGC-1α; PPARγ coactivator 1α; PPARγ; peroxisome proliferator-activated receptor γ; PIM; protein import machinery; RBPs; RNA-binding proteins; rho; ⁻; cells; cells with partially depleted mtDNA; rho; ⁰; cells; cells with completely depleted mtDNA; ROS; reactive oxygen species; SAM; sorting and assembly (SAM); SCO2; synthesis of cytochrome; c; oxidase 2; Sp1; specificity protein-1; SS; subsarcolemmal; STR; slow-twitch red; Tfam; mitochondrial transcription factor A; TFB1M/2M; mitochondrial transcription factor B-1M/2M; TIGAR; TP53-induced glycolysis and apoptosis regulator; TIM; translocase of the inner membrane; TOM; translocase of the outer membrane; TPR; tetratricopeptide repeat; USF-1; upstream stimulatory factor-1Subsarcolemmal; Intermyofibrillar; PGC-1; Tfam; p53; Signaling; Protein import; RNA stability; Plasticity

Mitochondrial death effectors: Relevance to sarcopenia and disuse muscle atrophy by Emanuele Marzetti ⁎; Judy C.Y. Hwang; Hazel A. Lees; Stephanie E. Wohlgemuth; Esther E. Dupont-Versteegden; Christy S. Carter; Roberto Bernabei; Christiaan Leeuwenburgh ⁎ (pp. 235-244).

Accelerated apoptosis in skeletal muscle is increasingly recognized as a potential mechanism contributing to the development of sarcopenia of aging and disuse muscle atrophy. Given their central role in the regulation of apoptosis, mitochondria are regarded as key players in the pathogenesis of myocyte loss during aging and other atrophying conditions. Oxidative damage to mitochondrial constituents, impaired respiration and altered mitochondrial turnover have been proposed as potential triggering events for mitochondrial apoptotic signaling. In addition, iron accumulation within mitochondria may enhance the susceptibility to apoptosis during the development of sarcopenia and possibly acute muscle atrophy, likely through exacerbation of oxidative stress. Mitochondria can induce myocyte apoptosis via both caspase-dependent and independent pathways, although the apoptogenic mediators involved may be different depending on age, muscle type and specific atrophying conditions. Despite the considerable advances made, additional research is necessary to establish a definite causal link between apoptotic signaling and the development of sarcopenia and acute atrophy. Furthermore, a translational effort is required to determine the role played by apoptosis in the pathogenesis of sarcopenia and disuse-induced muscle loss in human subjects.

Keywords: Mitochondria; Iron; Sarcopenia; Muscle atrophy; Apoptosis; Caspases; Endonuclease G; Apoptosis-inducing factor

Functional complementation of mitochondrial DNAs: Mobilizing mitochondrial genetics against dysfunction by Eric A. Schon; Robert W. Gilkerson (pp. 245-249).

Human mitochondrial DNA (mtDNA) is a 16.6-kb circular genome that is typically found in ∼1000 copies per cell. Frequently, one or more forms of mtDNA (i.e. wildtype (WT) and one or more mutant variants) will co-exist within an individual cell, a situation termed heteroplasmy; however, it has been unclear how different mitochondria and mtDNA populations interact functionally in a heteroplasmic cell system. Using sequence-specific microscopic methods to examine mtDNA at suborganellar resolution, we examined the submitochondrial organization of mtDNA heteroplasmy in nucleoids, the DNA–protein complexes that organize and package mtDNA. Our recent results reveal that, while heterologous mtDNAs are generally maintained stably in separate nucleoid populations, the two mtDNAs transcomplement each other to restore WT-like levels of mitochondrial function and morphology. These findings reveal that the diffusion of mtDNA-derived transcripts through the mitochondrial matrix allows for transcomplementation, despite the apparent genetic autonomy of nucleoids. The fundamental ability of mtDNAs to complement each other within the matrix of the mitochondrial network provides a mechanistic basis for therapeutic strategies designed to restore mitochondrial function in heteroplasmic cells by increasing WT mtDNA content, particularly in light of the emerging connection between the processes of mitochondrial fission/fusion and mtDNA nucleoid organization.

Keywords: Mitochondria; Mitochondrial DNA; Nucleoid; Complementation

Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle by Hu Ding; Ning Jiang; Huijun Liu; Xiaoran Liu; Danxia Liu; Fei Zhao; Li Wen; Shusen Liu; Li Li Ji; Yong Zhang (pp. 250-256).

The purpose of this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and mitochondrial energy metabolism in response to altered energy demand during prolonged exercise in rat skeletal muscle. Male Sprague–Dawley rats were subjected to an acute bout of treadmill running at various durations and killed immediately or during recovery. Mfn1/2 and Fis1 mRNA and protein contents, reactive oxygen species (ROS) generation, state 3 and state 4 respiration rates, trans-innermembrane potential and ATP synthase activity were measured in isolated muscle mitochondria. We found that (1) Mfn1/2 mRNA contents were progressively decreased during 150 min of exercise, along with decreased Mfn 1 protein levels. Fis1 mRNA and protein contents showed significant increases after 120–150 min of exercise. These changes persisted through the recovery period up to 24 h. (2) Mitochondrial ROS generation and state 4 respiration showed progressive increases up to 120 min, but dropped at 150 min of exercise. (3) State 3 respiration rate and respiratory control index were unchanged initially but decreased at 150 and 120 min of exercise, respectively, whereas ATP synthase activity was elevated at 45 min and returned to resting level thereafter. Our data suggested that the gene expression of mitochondrial fusion and fission proteins in skeletal muscle can respond rapidly to increased metabolic demand during prolonged exercise, which could significantly affect the efficiency of oxidative phosphorylation.

Keywords: Abbreviations; Mfn1/2; mitofusin1/2; Fis1; fission 1; Drp-1; dynamin-related protein-1; ROS; reactive oxygen species; RCI; respiration control indexExercise; Fusion; Fission; Mitochondria; Skeletal muscle; Rat

Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells by Chien-Tsun Chen; Shu-Han Hsu; Yau-Huei Wei (pp. 257-263).

Stem cell research has received increasing attention due to their invaluable potentials in the clinical applications to cure degenerative diseases, genetic disorders and even cancers. A great number of studies have been conducted with an aim to elucidate the molecular mechanisms involved in the regulation of self-renewal of stem cells and the mysterious circuits guiding them to differentiate into all kinds of progenies that can replenish the cell pools. However, little effort has been made in studying the metabolic aspects of stem cells. Mitochondria play essential roles in mammalian cells in the generation of ATP, Ca²⁺ homeostasis, compartmentalization of biosynthetic pathways and execution of apoptosis. Considering the metabolic roles of mitochondria, they must be also critical in stem cells. This review is primarily focused on the biogenesis and bioenergetic function of mitochondria in the differentiation process and metabolic features of stem cells. In addition, the involvement of reactive oxygen species and hypoxic signals in the regulation of stem cell pluripotency and differentiation is also discussed.

Keywords: Stem cell differentiation; Mitochondria; Metabolic shift; Reactive oxygen species (ROS); Warburg effect

Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer by Wen-Yi Hung; Chew-Wun Wu; Pen-Hui Yin; Chun-Ju Chang; Anna Fen-Yau Li; Chin-Wen Chi; Yau-Huei Wei; Hsin-Chen Lee ⁎ (pp. 264-270).

Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers.Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed.We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells.Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.

Keywords: Somatic mutation; mtDNA; Gastric cancer; Drug resistance; Migration

Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions by Zhinan Ding; Jingzhang Ji; Guorong Chen; Hezhi Fang; Shihui Yan; Lijun Shen; Jia Wei; Kaiyan Yang; Jianxin Lu ⁎; Yidong Bai ⁎ (pp. 271-274).

Mitochondrial defects have been associated with various human conditions including cancers.We analyzed the mutations at the mitochondrial DNA (mtDNA) in patients with different thyroid lesions. In particular, in order to investigate if the accumulation of mtDNA mutations play a role in tumor progression, we studied the highly variable main control region of mtDNA, the displacement-loop (D-loop) in patients with non-tumor nodular goiters, with benign thyroid adenomas, and with malignant thyroid carcinomas. Total thyroid tumor or goiter samples were obtained from 101 patients, matched with nearby normal tissue and blood from the same subject.Noticeably, mitochondrial microsatellite instability (mtMSI) was detected in 2 of 19 nodular goiters (10.53%), and 8 of 77 (10.39%) malignant thyroid carcinomas. In addition, 6 patients, including 5 (6.49%) with malignant thyroid carcinomas and 1 (5.26%) with nodular goiter, were found to harbor point mutations. The majority of the mutations detected were heteroplasmic.Our results indicate that mtDNA alterations in the D-loop region could happen before tumorigenesis in thyroid, and they might also accumulate during tumorigenesis.

Keywords: Mitochondrial DNA mutation; Mitochondrial microsatellite instability; D-loop region; Thyroid; Tumor progression

Mitochondrial dysfunction enhances the migration of vascular smooth muscles cells via suppression of Akt phosphorylation by Sun Young Ahn; Yon-Sik Choi; Hyun-Jung Koo; Jae Hoon Jeong; Wook Ha Park; Minseok Kim; Ying Piao; Youngmi Kim Pak (pp. 275-281).

Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues.We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC).The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC.Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease.Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis.

Keywords: Abbreviations; VSMC; vascular smooth muscle cells; oxLDL; oxidized low density lipoprotein; ddC; dideoxycytidine; OXPHOS; oxidative phosphorylation; AMPK; AMP-activated protein kinase; TCN; triciribine; AICAR; aminoimidazole carboxamide ribonucleotide; OCR; oxygen consumption rate; SFM; serum free mediaVSMC; Insulin signaling; Mitochondrial dysfunction; Atherosclerosis; Akt/PKB; oxLDL

Mitochondrial dysfunction and metabolic syndrome—looking for environmental factors by Hong Kyu Lee; Young Min Cho; Soo Heon Kwak; Soo Lim; Kyong Soo Park; Eun Bo Shim (pp. 282-289).

The centerpiece of the pathophysiologic mechanism of metabolic syndrome is insulin resistance. Recently, it is becoming evident that mitochondrial dysfunction is closely related to insulin resistance and metabolic syndrome. The underlying mechanism of mitochondrial dysfunction is very complex, which includes genetic factors from both nuclear and mitochondrial genome and numerous environmental factors. Several mitochondrial DNA polymorphisms are associated with the components of metabolic syndrome. Numerous chemicals and drugs may cause mitochondrial dysfunction and insulin resistance. Notably, it was recently reported that serum levels of several mitochondrial toxins, such as persistent organic pollutants are associated with metabolic syndrome, which necessitates further investigation to reveal its precise mechanism. Given that the health impact of metabolic syndrome is tremendous, it is necessary to develop therapeutic modalities to correct mitochondrial dysfunction or at least to halt its aggravation. In this regard, exercise can improve both mitochondrial function and insulin sensitivity, and some pharmaceutical agents were reported to improve mitochondrial function. However, further studies are warranted to find more effective therapeutic strategies to treat mitochondrial dysfunction. By doing so, we can also shed light on the path of research for other diseases related to mitochondrial dysfunction.

Keywords: Insulin resistance; Metabolic syndrome; Mitochondrial dysfunction; Persistent organic pollutant; Scaling law; Type 2 diabetes mellitus

Mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular degeneration by Ming-Jen Hsu; Joen-Rong Sheu; Chien-Huang Lin; Ming-Yi Shen; Chung Y. Hsu (pp. 290-296).

Prevailing evidence suggests that amyloid beta peptide (Aβ), a key mediator in age-dependent neuronal and cerebrovascular degeneration, activates death signaling processes leading to neuronal as well as non-neuronal cell death in the central nervous system. A major cellular event in Aβ-induced death of non-neuronal cells, including cerebral endothelial cells, astrocytes and oligodendrocytes, is mitochondrial dysfunction. The death signaling cascade upstream of mitochondria entails Aβ activation of neutral sphingomyelinase, resulting in the release of ceramide from membrane sphingomyelin. Ceramide then activates protein phosphatase 2A (PP2A), a member in the ceramide-activated protein phosphatase (CAPP) family. PP2A dephosphorylation of Akt and FKHRL1 plays a pivotal role in Aβ-induced Bad translocation to mitochondria and transactivation of Bim. Bad and Bim are pro-apoptotic proteins that cause mitochondrial dysfunction characterized by excessive ROS formation, mitochnondrial DNA (mtDNA) damage, and release of mitochondrial apoptotic proteins including cytochrome c, apoptosis inducing factor (AIF), endonuclease G and Smac. The cellular events activated by Aβ to induce death of non-neuronal cells are complex. Understanding these death signaling processes will aid in the development of more effective strategies to slow down age-dependent cerebrovascular degeneration caused by progressive cerebrovascular Aβ deposition.

Keywords: Aging; Amyloid beta peptide; Apoptosis; Ceramide; Cerebrovascular disease; Endothelial cells; Mitochondria; Stroke

Impaired mitochondrial bioenergetics determines glutamate-induced delayed calcium deregulation in neurons by Andrey Y. Abramov; Michael R. Duchen (pp. 297-304).

Accumulation of glutamate in ischaemic CNS is thought to amplify neuronal death during a stroke. Exposure of neurons to toxic glutamate concentrations causes an initial transient increase in [Ca²⁺]_c followed by a delayed increase commonly termed delayed [Ca²⁺]_c deregulation (DCD).We have used fluorescence imaging techniques to explore differences in glutamate-induced DCD in rat hippocampal neurons after different periods of time in culture (days in vitro; DIV).The amplitude of both the initial [Ca²⁺]_c signal and the number of cells showing DCD in response to glutamate increased with the duration of culture. The capacity of mitochondria to accumulate calcium in permeabilised neurons decreased with time in culture, although mitochondrial membrane potential at rest did not change. The rate of ATP consumption, measured as an increase in [Mg²⁺]_c following inhibition of ATP synthesis, was lower in ‘young’ neurons. The sensitivity of ‘young’ neurons to glutamate-induced DCD approximated to that of ‘old’ neurons when mitochondrial function was impaired using either FCCP or oligomycin. Further, following such treatment, cells showed a DCD-like response to increased [Ca²⁺]_c induced by KCl induced depolarisation which was never otherwise seen.Thus, changes in cellular bioenergetics dictate the onset of DCD in response to glutamate.

Keywords: Mitochondria; Glutamate; Excitotoxicity; Calcium

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation by Jia Qu; Xiangtian Zhou; Fuxin Zhao; Xiaoling Liu; Minglian Zhang; Yan-Hong Sun; Min Liang; Meixia Yuan; Qi Liu; Yi Tong; Qi-Ping Wei; Li Yang; Min-Xin Guan (pp. 305-312).

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families.One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA).Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families.The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families.

Keywords: Mitochondria; Leber's hereditary optic neuropathy; Penetrance; Haplogroup; Mutation; Chinese; ND6

Pyruvate therapy for Leigh syndrome due to cytochrome c oxidase deficiency by Hirofumi Komaki; Yutaka Nishigaki; Noriyuki Fuku; Hiroko Hosoya; Kei Murayama; Akira Ohtake; Yu-ichi Goto; Hiroyuki Wakamoto; Yasutoshi Koga; Masashi Tanaka (pp. 313-315).

Recently we proposed the therapeutic potential of pyruvate therapy for mitochondrial diseases. Leigh syndrome is a progressive neurodegenerative disorder ascribed to either mitochondrial or nuclear DNA mutations.In an attempt to circumvent the mitochondrial dysfunction, we orally applied sodium pyruvate and analyzed its effect on an 11-year-old female with Leigh syndrome due to cytochrome c oxidase deficiency accompanied by cardiomyopathy. The patient was administered sodium pyruvate at a maintenance dose of 0.5 g/kg/day and followed up for 1 year.The exercise intolerance was remarkably improved so that she became capable of running. Echocardiography indicated improvements both in the left ventricle ejection fraction and in the fractional shortening. Electrocardiography demonstrated amelioration of the inverted T waves. When the pyruvate administration was interrupted because of a gastrointestinal infection, the serum lactate level became elevated and the serum pyruvate level, decreased, suggesting that the pyruvate administration was effective in decreasing the lactate-to-pyruvate ratio.These data indicate that pyruvate therapy was effective in improving exercise intolerance at least in a patient with cytochrome c oxidase deficiency.Administration of sodium pyruvate may prove effective for other patients with cytochrome c oxidase deficiency due to mitochondrial or nuclear DNA mutations.

Keywords: Pyruvate therapy; Leigh syndrome; Cytochrome; c; oxidase deficiency; Exercise intolerance; Lactate-to-pyruvate ratio

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis by Mikael Widersten; Ann Gurell; Diana Lindberg (pp. 316-326).

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.

Keywords: Epoxide hydrolase; Structure–activity relationship; Mechanism; Stereospecificity; Biocatalysis; New enzymes; Directed evolution

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala→Val] by Barbara Manconi; Maria Cristina De Rosa; Maria Pia Cappabianca; Alessandra Olianas; Cristiana Carelli Alinovi; Fabrizio Mastropietro; Donatella Ponzini; Antonio Amato; Mariagiuseppina Pellegrini (pp. 327-335).

Haemoglobin Roma [β115(G17)Ala→Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala→Val substitution at the α1β1 interface.The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a G CC→G TC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.This study suggests that the β115(G17)Ala→Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.

Keywords: Abbreviations; Hb; hemoglobin; RP-HPLC; reversed phase-HPLC; nt; nucleotide; ESI; electrospray ionization; TFA; trifluoroacetic acid; MS/MS; Tandem Mass; XIC; extracted ion current; BPG; 2,3-bisphosphoglycerate; P; ₅₀; partial pressure of O; ₂; required to saturate 50% of the hemes; Hb HK; Hb Hradek KraloveHaemoglobin variant; Codon 115 β globin gene; α1β1 interface; Oxygen affinity; Homology modeling

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage by Tricia A. Ulmer; Vicki Keeler; Andre Sabine André; Hans-Joachim Gabius; Lambert Loh; Laferte Suzanne Laferté (pp. 336-343).

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.Proteolytic cleavage of TAA90K may have functional implications in colon cancer.

Keywords: Abbreviations; APMA; p; -aminophenylmercuric acetate; BSA; bovine serum albumin; CyCAP; cyclophilin C-associated protein; DMEM; Dulbecco's minimum essential medium; ECM; extracellular matrix; EDTA; ethylenediaminetetraacetic acid; ELISA; enzyme-linked immunosorbent assay; FBS; fetal bovine serum; HPLC; high-performance liquid chromatography; IgG; immunoglobulin G; IL-1β; interleukin-1β; IL-6; interleukin-6; MEM; minimal essential medium; MMP; matrix metalloproteinase; PBS; phosphate buffered saline; PBST; PBS/04% Tween 20; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TAA90K; tumor-associated antigen 90KColon cancer; Galectin-3; Matrix metalloproteinases; Tumor-associated antigen 90K

Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells by Jane Sun; Dingyan Wang; Tianru Jin (pp. 344-351).

Epidemiological and experimental evidence that support the correlation between Type 2 diabetes mellitus (T2D) and increased risks of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and canonical Wnt signaling pathways. Insulin was shown to stimulate Wnt target gene expression, utilizing the effector of the Wnt signaling pathway. Whether insulin affects expression of components of Wnt pathway has not been extensively examined.cDNA microarray was utilized to assess the effect of insulin on gene expression profile in the rat intestinal non-cancer IEC-6 cell line, followed by real-time RT-PCR, Western blotting and reporter gene analyses in intestinal cancer and non-cancer cells.Insulin was shown to alter the expression of a dozen of Wnt pathway related genes including TCF-4 (=TCF7L2) and frizzled- (Fzd-4). The stimulatory effect of insulin on TCF-4 expression was then confirmed by real-time RT-PCR, Western blotting and luciferase reporter analyses, while the activation on Fzd-4 was confirmed by real-time PCR.Our observations suggest that insulin may crosstalk with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.

Keywords: Insulin; Wnt; TCF-4; c-Myc; Fzd-4; cDNA microarray

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling by Ji-Hye Yoon; Soo-A Kim; Seong-Min Kwon; Jong-Hwan Park; Hee-Sae Park; Yong-Chul Kim; Jung-Hoon Yoon; Sang-Gun Ahn (pp. 352-358).

5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified.Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis.5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis.These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling.This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.

Keywords: Abbreviations; 5′-NIO; 5′-nitro-indirubinoxime; Hes-1; hairy enhancer of split-1; CDK; cyclin-dependent kinase; MAPK; mitogen-activated protein kinase; GSK3β; glycogen kinase 3β; MMP; matrix metalloproteinase; VEGF; vascular endothelial growth factor; PARP; poly(ADP-ribose)polymerase; Cox; cyclooxygenase5′-NIO; Notch-1; Notch-3; Salivary gland adenocarcinoma; Apoptosis

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes by Kadapakkam Nandabalan Sangeetha; Sundaresan Sujatha; Velusamy Shanmuganathan Muthusamy; Singaravel Anand; Nirmal Nithya; Devadasan Velmurugan; Arun Balakrishnan; Baddireddi Subhadra Lakshmi (pp. 359-366).

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.

Keywords: Abbreviations; EAE; Ethyl acetate extract; IRβ; insulin receptor β; IRS; insulin receptor substrate; IRTK; insulin receptor tyrosine kinase; PI3K; phosphotidyl inositol 3-kinase; PKB; protein kinase B; GLUT4; glucose transporter; GSK3 β; Glycogen synthase kinase 3β; NBT; Nitro blue tetrazolium chloride; BCIP; 5-Bromo-4-chloro-3-indolylphosphate; MTT-3-(4,5-Dimethylthiazol-2-yl)-2,5; diphenyltetrazolium bromide; DMEM; Dulbecco's Modified Eagles medium; PTP1B; protein tyrosine phosphatase 1B; GS; glycogen synthesis; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase; NIDDM; Non insulin dependent diabetes mellitus; RT-PCR; reverse transcription polymerase chain reaction; LDH; lactate dehydrogenase; pNPP; p-nitrophenyl phosphate; 2-DOG; 2-deoxy-D-; ³; [1-H]-glucose uptake; WT; wortmannin; Gen; genistein; NMR; nuclear magnetic resonance spectroscopy3β-Taraxerol; Mangifera indica; Glycogen synthesis; Glucose uptake; 3T3-L1 adipocytes; GSK3β; PI3K

Selective binding interactions of deramciclane to the genetic variants of human α₁-acid glycoprotein by Ilona Fitos; Júlia Visy; Miklós Simonyi; György Mády; Ferenc Zsila (pp. 367-372).

α₁-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP.The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis.DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER.DER has high-affinity binding ( K_a≥2×10⁶ M^-1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored.The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.

Keywords: Abbreviations; AGP; α; ₁; -Acid glycoprotein; AMI; amitriptyline; ANS; 8-anilino-1-naphtalene-sulfonic acid; AO; acridine orange; AODB; acridine orange-10-dodecyl bromide; CD; circular dichroism; CPZ; chlorpromazine; DER; deramciclane; DIC; dicumarol; DIS; disopyramide; IMI; imipramine; MIF; mifepristone; PRO; propranololα; ₁; -Acid glycoprotein; Genetic variant; Deramciclane; Allosteric binding interaction; Induced circular dichroism; Chiral conformation; Dicumarol

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle by Alhondra Solares-Pérez; Jorge A. Sánchez; Alejandro Zentella-Dehesa; María C. García; Ramón M. Coral-Vázquez (pp. 373-379).

δ-Sarcoglycan (δ-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca²⁺ permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease.Isolated skeletal muscle fibers of wild-type and δ-SG KO mice were used to investigate whether the absence of δ-SG alters the increase in intracellular Ca²⁺ during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca²⁺ transient recording techniques with fluorescent indicators were used.Ca²⁺ transients during single twitches and tetani generated by muscle fibers of δ-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca²⁺ and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a δ-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles.δ-SG has a role in calcium homeostasis in skeletal muscle fibers.These results support a possible role of δ-SG on calcium homeostasis. The alterations caused by the absence of δ-SG may be related to the pathogenesis of muscular dystrophy.

Keywords: Abbreviations; δ-SG; δ-Sarcoglycan; KO; knockout; DGC; dystrophin-glycoprotein complex; SG–SSPN; sarcoglycan–sarcospan; SG; sarcoglycan; SR; sarcoplasmic reticulum; EDL; extensor digitorum longus; SOC; store-operated calcium channelsMuscular dystrophy; Sarcoglycan–sarcospan complex; Ca; ²⁺; transients; Sarcoplasmic reticulum; Muscle fibers; Intracellular calcium

Dicer knockdown induces fibronectin-1 expression in HEK293T cells via induction of Egr1 by Kai-Fu Tang; Guan-Bin Song; Yi-Song Shi; Lin Yuan; Yong-Hua Li (pp. 380-384).

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells.siRNA transfection, cell migration assay, real-time RT–PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V–FITC assay were utilized.Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells.Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner.Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.

Keywords: Fibronectin-1; Dicer; Egr1; Cell migration

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches by Xiantao Li; Markus Rapedius; Thomas Baukrowitz; Gong Xin Liu; D.K. Srivastava; Jürgen Daut; Peter J. Hanley (pp. 385-391).

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5‑HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.5-HD inhibited (IC₅₀∼30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.

Keywords: K; _ATP; channel; Preconditioning; Single-channel current; Myocyte

Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering by M.J. Clarke; J.B. Artero; M. Moulin; P. Callow; J.A. Carver; P.C. Griffiths; M. Haertlein; J.J. Harding; K.M. Meek; P. Timmins; J.W. Regini (pp. 392-397).

α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α-crystallin (α_H) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α_H in D₂O incubated at 65 °C increased from 69±3 to 81±5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H₂O buffer (γE_D/H₂O) and hydrogenous γE-crystallin in D₂O buffer (γE_H/D₂O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α_H complexes. The evolution of the aggregation size/shape as an indicator of α_H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α_H, γE_H, and γE_D. It was found that Rg(α_H:γE_D/D₂O)>Rg(α_H:γE_H/D₂O)>Rg(α_H/D₂O) and that Rg(α_H:γE_H/D₂O)≈Rg(α_H/D₂O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action.

Keywords: Crystallin; Chaperone; Neutron scattering; X-ray scattering; Heat shock protein

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases by Henry K. Bayele; Paul J. Murdock; K. John Pasi (pp. 398-404).

Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood.We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay.We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme.Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases.The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.

Keywords: Abbreviations; DsbA; d; efective in di; s; ulphide; b; ond formation; GSH; reduced glutathione; GSSG; oxidized glutathione; PDI; protein disulphide isomerase; TNB; thionitrobenzoic acid; Trx; thioredoxin; sRNase A; scrambled ribonuclease ABlood coagulation; Protein folding; Oxidoreductase; Peptide mimetic; Redox catalyst; Cofactor; Tenase complex

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin by Hiroshi Shiraishi; Hideaki Okamoto; Hiromitsu Hara; Hiroki Yoshida (pp. 405-415).

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.

Keywords: Abbreviations; Apaf1; apoptotic protease activating factor 1; NPCs; neural progenitor cells; ROS; reactive oxygen species; EGF; epidermal growth factor; CNS; central nervous system; NAC; N; -acetyl-L-cysteine; GFP; green fluorescent protein; LC3; microtubule-associated protein light chain 3; NPMM; neural progenitor maintenance medium; N2/DMEM/F12; Dulbecco's modified Eagle's medium/F12 containing N2 supplement; FGF; fibroblast growth factor; MEFs; mouse embryonic fibroblasts; HBSS; Hank's balanced salt solution; PI; propidium iodide; DAPI; 4′,6-diamidino-2-phenylindole; CM-H; ₂; DCFDA; 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; Nec-1; necrostatin-1; BHA; 3(2)-; t; -butyl-4-hydroxyanisole; RIP1; receptor interacting protein 1; MMP; mitochondrial membrane permeabilization; mPT; mitochondrial permeability transition; NMDA; N-methyl-D-aspartate; TNF-α; tumor necrosis factor alphaApaf1; Neural progenitor cell; Necrosis-like cell death; Necroptosis

Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal-responsive genes by Zoia R. Stoytcheva; Vladimir Vladimirov; Vanessa Douet; Ilko Stoychev; Marla J. Berry (pp. 416-424).

Selenoprotein H is a redox-sensing DNA binding protein that upregulates genes involved in antioxidant responses. Given the known links between oxidative stress and heavy metals, we investigated the potential for regulation of selenoprotein H by metals. In silico analysis of the selenoprotein H genes from nine species reveals multiple predicted metal response elements (MREs). To validate MRE function, we investigated the effects of zinc or cadmium addition and metal-responsive transcription factor 1 (MTF-1) knockout on selenoprotein H mRNA levels. Chromatin immunoprecipitation was used to directly assess physical binding of the transcription factor to MREs in the human and mouse selenoprotein H genes. The results reported herein show that selenoprotein H is a newly identified target for MTF-1. Further, whereas nearly all prior studies of MREs focused on those located in promoters, we demonstrate binding of MTF-1 to MREs located downstream of the transcription start sites in the human and murine selenoprotein H genes. Finally, we identified MREs in downstream sequences in 15 additional MTF-1 regulated genes lacking promoter MREs, and demonstrated MTF-1 binding in three of these genes. This regulation via sequences downstream of promoters highlights a new direction for identifying previously unrecognized target genes for MTF-1.

Keywords: Abbreviations; MRE; metal responsive element; MTF-1; metal element binding transcription factor 1; TrSS; translation start site; TSS; transcription start siteSelenoprotein H; MTF-1; MREs

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BBA - General Subjects (v.1800, #3)