Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Effect of diets supplemented with different conjugated linoleic acid (CLA) isomers on protein expression in C57/BL6 mice

Background

The individual genetic variations, as a response to diet, have recently caught the attention of several researchers. In addition, there is also a trend to assume food containing beneficial substances, or to supplement food with specific compounds. Among these, there is the conjugated linoleic acid (CLA), which has been demonstrated to reduce fat mass and to increase lean mass, even though its mechanism of action is still not known. We investigated the effect of CLA isomers (CLA c9,t11 and CLA t10,c12) on the proteomic profile of liver, adipose tissue, and muscle of mouse, with the aim of verifying the presence of a modification in fat and lean mass, and to explore the mechanism of action.

Methods

C57/BL6 mice were fed for 2 months with different diets: (1) standard chow, (2) CLA c9,t11 diet, (3) CLA t10,c11 diet, (4) CLA isomers mixture diet, and (5) linoleic acid diet. The proteomic profile of liver, white adipose tissue, and muscle was investigated. Statistical significance of the spots with an intensity higher than twofold in expression compared to the control was tested using student’s t test (two-tail).

Results

We found that both isomers modulate the proteomic profiles of liver, adipose tissue, and muscle by different mechanisms of action. Liver steatosis is mostly due to the isomer CLA t10,c12, since it alters the expression of lipogenetic proteins; it acts also reducing the adipose tissue and increasing fatty acid oxidation in muscle. Conversely, CLA c9,t11 has no relevant effects on liver and adipose tissue, but acts mostly on muscle, where it enhances muscular cell differentiation.

Conclusions

Administration of CLA in humans has to be carefully personalized, since even considering the presence of a species-specific effect, adverse effects might occur on long-term supplementation. Here we demonstrated that, in mouse, CLA is effective in reducing fat mass, but it also induces liver steatosis. The increase of lean mass is linked to an induction of cell proliferation, which, on long-term supplementation, might also lead to adverse effects.

     

High-pass filtering of input signals by the Ih current in a non-spiking neuron, the retinal rod bipolar cell

Hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels mediate the I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of I(h) in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated-patch recordings in the dark-adapted slice found that RBCs exhibit I(h), and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency-modulated current stimuli (0.1-30 Hz), displays band-pass behavior in the range of I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), in combination with low-pass filtering by passive properties, fully accounts for this frequency-tuning. Correcting for the depolarization introduced by shunting through the pipette-membrane seal, leads to predict that in darkness I(h) is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1-2, in combination with markers of RBCs (PKC) and rod-RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.     

Bait traps remain attractive to euglossine bees even after two weeks: a report from Brazilian Atlantic forest

Bait traps are effective and commonly used method in the studies of orchid bees. Important questions in the context of this method, including those related to bait dispersion, how long baits remain attractive, the distance from which males are supposed to be attracted to lures and so on, are still open subjects. Data on the attractiveness of bait traps that have remained in the field during two weeks in a large Atlantic forest preserve are presented. Four main results emerge from the data: (i) the abundance of specimens collected per day decreased in all the attractants as the traps were left on the field; (ii) despite this decrease, the absolute number of individuals collected after eight and fifteen days is remarkably, mostly in eugenol and vanillin baits; (iii) the vast majority of species, 22 of 25, was already collected on the first sample day; (iv) a consistent variation in the relative abundance of individuals collected in each scent as collections were made. We urge that bait traps should not be left in the field beyond what is strictly necessary since there is a real possibility of collecting a significant number of individuals as these traps remain available.     

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Control of polymerization by J chain

Cell suspensions of mouse plasma-cell tumours secreting IgA (immunoglobulin A) and IgM (immunoglobulin M) were incubated with radioactive leucine for various periods of time. The secreted immunoglobulins were precipitated from the culture medium with specific rabbit antisera to determine the relative distribution of radioactivity among the different molecular species, and to estimate the fraction of total radioactivity in the J chain. For IgM-secreting cells there is a balanced synthesis of 7S subunits and J chains, and the secreted product is uniformly assembled to the pentamer. In cells secreting IgA, however, the results demonstrate that the pool of intracellular J chain is less than the intracellular IgA pool. The concentration of J chain is therefore limiting and is less than the requirement for complete polymerization. The major factor that determines whether an intracellular monomer is secreted as such or is polymerized with the addition of J chain is therefore the amount of intracellular J chain. When this is limiting, as it is in cells secreting IgA, then monomer will be secreted.