The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria

The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.     

Bone tissue changes in rats during prolonged restriction of motor activity

The objective of this investigation was to measure the effect of prolonged restriction of motor activity (hypokinesia) of rats on the mass, density, mineral composition, reconstruction parameters and elemental composition of their bone tissue. The studies were done during 90 days of hypokinesia (HK) on 90 male Wistar rats equally divided into two groups: (1) vivarium control rats (VCR) and (2) hypokinetic rats (HKR). For the simulation of the hypokinetic effect the HKR group was kept for 90 days in small individual cages made of wood that restricted the movements of rats in all directions without hindering food and water intakes. During the prehypokinetic period of 15 days and during the hypokinetic period of 90 days bone mass, bone density, bone calcium and phosphorus concentrations, bone reconstruction parameters and elemental composition of bones were determined. During the same periods food intake and body weight losses were also measured. In the HKR group signs of osteoporosis in the spongy structures of the tubular bones were observed; they also showed significant decrease in rat femur weight, and in cross section of the rat femur, and in mineral concentrations of the femoral head when compared with the VCR group. The HKR group also show a significant decrease in food intake and body weight when compared with the VCR group. The corresponding parameters did not change significantly in the VCR group when compared with the baseline control values. It was concluded that prolonged exposure to HK induced osteoporosis and structural changes in bones. This apparently occurred due to inhibition of bone tissue formation in the HKR group.     

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.     

Daily magnesium supplementation on serum and urinary magnesium changes in rats during prolonged restriction of motor activity

The objective of this investigation was to determine whether a plentiful magnesium (Mg²⁺) supplementation might be used to normalize or prevent Mg deficiency. This is manifested by increased rather than decreased serum Mg²⁺ concentration as is observed during prolonged hospitalization, which is developed during prolonged hypokinesia (HK) (decreased motor activity). Eighty male Wistar rats with an initial body weight of 370–390 g were used to perform the studies: They were equally divided into four groups:

1. Unsupplemented control animals (UCA);
2. Supplemented control animals (SCA);
3. Unsupplemented hypokinetic animals (UHA); and
4. Supplemented hypokinetic animals (SHA).

For the simulation of the hypokinetic effect, the hypokinetic animals were kept in small individual cages made of wood, which restricted their movements in all directions without hindering food and water intake. The control and hypokinetic supplemental animals receive 0.9 mg/mL Mg sulfate daily with their drinking water. Prior to and during the experimental period, urinary excretions of Mg, calcium, and phosphate along with their concentrations in serum, water intake, and urine excretion, and body weight were determined in the control and hypokinetic animals. In the supplemental and unsupplemental hypokinetic rats, urinary excretions and serum concentrations of electrolytes increased significantly, whereas serum concentration and urinary excretion thereof remained unchanged in the supplemented and unsupplemented control animals. It was concluded that a daily intake of large amounts of Mg supplementation cannot be used to prevent or normalize Mg deficiency in rats during prolonged exposure to HK.     

Quantitative changes in the content of nucleic acid bases and their derivatives in a culture ofPenicillium sizowi

Acid-soluble (“free”) nucleotides, nucleosides and bases were analyzed in the mycelium and in the culture filtrate of the fungusPenicillium sizowi, using micro-thin-layer chromatography on alumina and densitometry of the zones of the individual components. It was found that the levels of the various components underwent complicated changes, the corresponding curves exhibiting from one to three maxima which occur at different periods of cultivation. It was observed that a substantial amount of nucleotides, nucleosides and bases occurs in the medium as early as at the beginning of the exponential phase of growth. An attempt was made to elucidate some peculiarities of the nucleotide pool ofPenicillium species, using enzymes responsible for the individual transformations of nucleotides, nucleosides and bases.     

Ab initio molecular dynamics of the reactivity of vitamin C toward hydroxyl and $$ {HO}_2/{O}_2^{-} $$ radicals

Vitamin C is one of the most abundant exogenous antioxidants in the cell, and it is of the utmost importance to elucidate its mechanism of action against radicals. In this study, the reactivity of vitamin C toward OH and \( {HO}_2/{O}_2^{-} \) radicals in aqueous medium was analyzed by ab initio molecular dynamics using CPMD code. The simulations led to results similar to those of static studies or experiments for the pair of \( {HO}_2/{O}_2^{-} \) radicals but bring new insights for the reactivity with hydroxyl radical: the reaction takes place before the formation of an adduct and consists of two steps: first an electron is transferred to hydroxyl radical and then the ascorbyl radical loses a proton.

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Graphical Abstract Reactivity of vitamin C toward hydroxyl and \( {HO}_2/{O}_2^{-} \) radicals

     

Conformational analysis of macrocyclic frankincense (<Emphasis Type="Italic">Boswellia</Emphasis>) diterpenoids

Frankincense oleoresin has been used in traditional medicine for more than 5000 years. The phytochemistry of frankincense (Boswellia spp.) resins includes triterpenoids (including boswellic acids and their derivatives), diterpenoids (cembrenoids and cneorubenoids), and essential oils. The macrocyclic cembrene diterpenoids may play a part in the biological activities of frankincense resin, but neither the biological targets nor the modes of interaction with the targets are currently known. How these macrocycles interact with biological macromolecules likely depends on what conformation(s) are energetically available to them. In this work, a conformational analysis of 15 Boswellia cembrene diterpenoids and 1 verticillane diterpenoid was carried out at the B3LYP/6-31G* and M06-2X/6-31G* levels of theory, including the SM8 aqueous solvation model. The lowest-energy conformations of boscartin B and incensole oxide were the same as the previously reported X-ray crystal structures, while the lowest-energy conformations of boscartins A and C were very similar to the crystal structures. Boscartins D-H and isoincensole oxide showed only one low-energy conformation for each compound and are predicted to be conformationally locked. Incensole, isoincensolol, and serratol are predicted to be conformationally mobile with several low-energy forms. The conformational mobility of Boswellia cembrenoid diterpenoids depends largely on the degree of epoxidation, either oxirane or tetrahydrofuran rings.     

Evidence of multiple introductions and genetic admixture of the Asian brush-clawed shore crab <Emphasis Type="Italic">Hemigrapsus takanoi</Emphasis> (Decapoda: Brachyura: Varunidae) along the Northern European coast

The Asian brush-clawed shore crab Hemigrapsus takanoi is a non-indigenous species along the Northern European coast. Although the history of range expansion of European H. takanoi has been well-documented, little is known about the genetic compositions of either the introduced European populations or the native Asian ones. We therefore collected H. takanoi broadly from their native Asian sites and introduced European ranges, and genotyped them by sequencing the mitochondrial 16S RNA gene and by analyzing nuclear microsatellite loci. Our results revealed that the H. takanoi Bay of Seine (France) populations consisted of a genetic admixture between populations in Japan and those in the Yellow Sea region. These French populations should be carefully monitored in the future, since the genetic admixture of multiple source populations may accelerate range expansion in non-indigenous organisms. Our results also suggested that shipping lines from East Asia were more probable vectors than historical juvenile oyster transportations from Japan for the foundation of present European H. takanoi populations. Interestingly, gene flow between populations in Japan and those in the Yellow Sea region (i.e., domestic invasion) was not observed despite the higher potential for artificial translocations via shipping lines in the native Asian range compared with those from Asia to Europe. The lack of domestic invasions implied that intra-specific priority effects of the resident H. takanoi populations played an important role in preventing the successful colonization of artificially-transferred individuals.     

Calcium signals regulate the functional differentiation of thymic iNKT cells

How natural or innate‐like lymphocytes generate the capacity to produce IL‐4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4⁺ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca²⁺) signals and continuing activity of the calcium release‐activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca²⁺ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca²⁺ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca²⁺ buffering in NKT2 cells. In addition, the low Ca²⁺ extrusion rate may also contribute to the higher Ca²⁺ level in NKT2 cells. Altogether, we identified ORAI‐dependent Ca²⁺ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.