Activation and inhibition of succinate-dependent Ca2+ transport in liver mitochondria during adaptation

Succinate-dependent Ca²⁺ accumulation was investigated in rat liver mitochondria and in operation biopsies of patients under states either with prevalence of adrenergic influences (administration of activating doses of adrenaline, acute phase of immobilization stress, initial period after allogenic transplantation, and acute phase of myocardial infarction) or with prevalence of the reciprocal mediator acetylcholine (late period after transplantation, chronic ulcer disease of stomach and duodenum). Adrenaline prevalence leads to increase of succinate-dependent ATP synthesis and Ca²⁺ accumulation, which is due to known activation of succinate oxidation. However, together with activation, inhibition of these processes was revealed. The inhibition phase prevails under chronic pathology. Therefore, reciprocal regulation of succinate oxidation and succinate-dependent Ca²⁺ transport in mitochondria occurs in the body in the course of adaptation. The reciprocal regulation of mitochondrial processes is considered as a mechanism of reciprocal regulation of physiological functions.     

Respiratory Chains from Aerobic Thermophilic Prokaryotes

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.     

Degradation of poly(tetramethylene succinate) by thermophilic actinomycetes

Various thermophilic actinomycetes were screened for their ability to degrade a high melting point, aliphatic polyester, poly(tetramethylene succinate) (PTMS), at 50 °C. By using the clear zone method, Microbispora rosea, Excellospora japonica and E. viridilutea were found to have PTMS-degrading activity. In a liquid culture with 100 mg PTMS film, M. rosea subsp. aerata IFO 14046 degraded about 50 mg film sample after 8 days. Degradation at the amorphous regions of the PTMS film was observed by scanning electron microscopy. This strain was also able to completely degrade poly(-caprolactone).     

The Quinol:Fumarate Oxidoreductase from the Sulphate Reducing Bacterium Desulfovibrio gigas: Spectroscopic and Redox Studies

The membrane bound fumarate reductase (FRD) from the sulphate-reducer Desulfovibrio gigas was purified from cells grown on a fumarate/sulphate medium and extensively characterized. The FRD is isolated with three subunits of apparent molecular masses of 71, 31, and 22 kDa. The enzyme is capable of both fumarate reduction and succinate oxidation, exhibiting a higher specificity toward fumarate (K _m for fumarate is 0.02 and for succinate 2 mM) and a reduction rate 30 times faster than that for oxidation. Studies by Visible and EPR spectroscopies allowed the identification of two B-type haems and the three iron–sulphur clusters usually found in FRDs and succinate dehydrogenases: [2Fe-2S]^2+/1+ (S1), [4Fe-4S]^2+/1+ (S2), and [3Fe-4S]^1+/0 (S3). The apparent macroscopic reduction potentials for the metal centers, at pH 7.6, were determined by redox titrations: –45 and –175 mV for the two haems, and +20 and –140 mV for the S3 and S1 clusters, respectively. The reduction potentials of the haem groups are pH dependent, supporting the proposal that fumarate reduction is associated with formation of the membrane proton gradient. Furthermore, co-reconstitution in liposomes of D. gigas FRD, duroquinone, and D. gigas cytochrome bd shows that this system is capable of coupling succinate oxidation with oxygen reduction to water.     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.     

Crystal structure of the RNA demethylase ALKBH5 from zebrafish

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N⁶-methyladenosine (m⁶A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m⁶A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m⁶A demethylation.     

Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5–100 μM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca²⁺ overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca²⁺ overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.     

Determination of dehydroandrographolide succinate in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS): Method development,validation and clinical pharmacokinetic study

A LC–MS/MS method was developed and validated for the determination of dehydroandrographolide succinate (DAS), a traditional Chinese medicine derivative used for the treatment of pneumonia, upper respiratory tract infection, and chronic bronchitis. Following protein precipitation, DAS was detected by ion transition at m/z 531.2/99.0 in multiple reaction monitoring mode with negative electrospray ionization-tandem mass spectrometry. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 10 ng/mL in human plasma. Good linearity was maintained over the range of 10–5000 ng/mL, and the correlation coefficient was better than 0.99. The accuracy ranged from 95.3% to 113%, RSD from 0.928% to 6.47%, for the within- and between-run analysis at all QC levels. The recovery ranged from 85.5% to 93.4% and the matrix effect from 107% to 119%. No significant carryover and good stability were found during method validation. The developed method was successfully applied to the determination of DAS in human plasma in a pharmacokinetic study following intravenous infusion of potassium sodium DAS to nine healthy volunteers.