Важливе історико-географічне дослідження

Рец. на кн. Темушева В.Н. "Гомельская земля в конце XV первой половине XVI в. Территориальные трансформации в пограничном регионе". — М.: "Квадрига", 2009. — 190 с.Review of the book: Temushev V.N. "Gomel Land in the Late 15th — the 1st half of the 16th Centuries. Territorial Transformations in the Frontier Area". — Moscow: "Kvadriga", 2009. — 190 p

The Non-Specific Drp1 Inhibitor Mdivi-1 Has Modest Biochemical Antioxidant Activity

Mitochondrial division inhibitor-1 (mdivi-1), a non-specific inhibitor of Drp1-dependent mitochondrial fission, is neuroprotective in numerous preclinical disease models. These include rodent models of Alzheimer’s disease and ischemic or traumatic brain injury. Among its Drp1-independent actions, the compound was found to suppress mitochondrial Complex I-dependent respiration but with less resultant mitochondrial reactive oxygen species (ROS) emission compared with the classical Complex I inhibitor rotenone. We employed two different methods of quantifying Trolox-equivalent antioxidant capacity (TEAC) to test the prediction that mdivi-1 can directly scavenge free radicals. Mdivi-1 exhibited moderate antioxidant activity in the 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS) assay. Half-maximal ABTS radical depletion was observed at ~25 μM mdivi-1, equivalent to that achieved by ~12.5 μM Trolox. Mdivi-1 also showed antioxidant activity in the α, α-diphenyl-β-picrylhydrazyl (DPPH) assay. However, mdivi-1 exhibited a reduced capacity to deplete the DPPH radical, which has a more sterically hindered radical site compared with ABTS, with 25 μM mdivi-1 displaying only 0.8 μM Trolox equivalency. Both assays indicate that mdivi-1 possesses biochemical antioxidant activity but with modest potency relative to the vitamin E analog Trolox. Future studies are needed to evaluate whether the ability of mdivi-1 to directly scavenge free radicals contributes to its mechanisms of neuroprotection

Magnesium sulfate protects against the bioenergetic consequences of chronic glutamate receptor stimulation.

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO4, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg(2+) (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO4 improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO4 or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO4 protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO4 protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate

Representative Type I (A, C) and Type II (B, D) bioenergetic profiles with or without MgSO₄ treatment.

<p>Neurons were pre-incubated for 1 hr in the absence or presence of MgSO₄ (3 or 10 mM). Three basal O₂ consumption rate (OCR) measurements were made in the continued absence or presence of MgSO₄, followed by control (con) or glutamate (glu, 100 µM) addition (first arrow). NMDA receptor activation was promoted by addition of the co-agonist glycine (10 µM) with glutamate. CNQX and MK801 (10 µM each) were added to end glutamate receptor stimulation, along with FCCP (3 µM) to simultaneously uncouple mitochondria and reveal respiratory capacity in the presence of endogenous substrates (second arrow). Excess mitochondrial substrate in the form of pyruvate (pyr, 10 mM) was then supplied to reveal maximal respiratory capacity (third arrow), followed finally by the complex III inhibitor antimycin A (AA, 1 µM) to inhibit mitochondrial O₂ consumption (fourth arrow). <b>(A)</b> and <b>(B)</b>. Absolute OCRs from Type I and Type II neurons, respectively. <b>(C)</b> and <b>(D)</b>. Baseline-normalized OCRs from Type I and Type II neurons, respectively. OCRs are normalized to the third baseline measurement prior to the addition of glutamate or control. Representative traces are mean ± SD from three wells. In some cases the error bars are smaller than the symbol size.</p

MgSO₄ pre-treatment preferentially enhances the bioenergetic response to glutamate in Type I neurons.

<p>Normalized <b>(A, B)</b> and absolute <b>(C, D)</b> O₂ consumption rates (OCRs) after glutamate receptor stimulation for Type I <b>(A, C)</b> and Type II <b>(B, D)</b> neuron classes are shown. OCRs in (A) and (B) are normalized to the control value corresponding to the first measurement after glutamate (glu) addition (i.e. the fourth measurement point, black circles, in the Fig. 1 traces). Results are mean ± SE from 21 (A, C) or 15 (B, D) independent experiments. *p<0.05 for glutamate plus Mg²⁺ with respect to glutamate alone (A, B). #p<0.05 for glutamate-treated with respect to control (A, B) or with respect to basal (C, D). †p<0.05 for Mg²⁺ (open bars) with respect to control (no MgSO₄, solid bars, C, D).</p

Pre-incubation with the mitochondrial substrate pyruvate mimics the beneficial bioenergetic effects of Mg²⁺ treatment.

<p>Normalized O₂ consumption rates (OCRs) after glutamate receptor stimulation for Type I neurons are shown in <b>(A)</b>. OCRs are normalized to the control value corresponding to the first measurement after glutamate addition as in Fig. 2A and B. Results are mean ± SE from 6 independent experiments. *p<0.05 relative to control or glutamate alone. #p<0.05 for glutamate plus pyruvate relative to glutamate plus Mg²⁺. Relative respiratory capacity in Type I neurons after glutamate receptor stimulation is shown in <b>(B)</b>. OCRs are normalized to the control value corresponding to the measurement just prior to antimycin A addition in the representative time course shown in Fig. 1 (i.e. at time 120 min, subsequent to the addition of both FCCP and pyruvate). Results are mean ± SE from 5 independent experiments. *p<0.05 relative to control or glutamate alone.</p

Chronic exposure of Type II neurons to 5 µM glutamate recapitulates a Type I bioenergetic phenotype.

<p><b>(A)</b> Type II neurons were pre-incubated for 1 hr in the absence or presence of 5 µM glutamate with the added absence or presence of MgSO₄ (3 mM) prior to measurement of OCRs. Treatments were also present throughout the OCR measurements. Glutamate (glu, 100 µM) or control (con) was added at the arrow. Numbers in the figure legend refer to the constitutively present (5 µM) or acutely added (100 µM) glutamate, respectively. OCRs are normalized to the third baseline measurement prior to the addition of glutamate or control. Representative traces are mean ± SD from three wells. <b>(B)</b> Mean normalized OCRs after glutamate receptor stimulation for the experiment depicted in (A). OCRs are normalized to the control value (black circles) corresponding to the first measurement after glutamate addition as in Fig. 2A and B. Results are mean ± SE from 3 independent experiments. *p<0.05 for the indicated comparisons. <b>(C)</b> Type II neurons were pre-incubated with or without 5 µM glutamate, 3 mM MgSO₄, or both, as in (A), prior to measurement of OCRs. In the presence of continued treatment, three basal OCR measurements were made, followed by the subsequent sequential additions of glutamate or control, FCCP+MK801+CNQX, pyruvate (pyr), and antimycin (AA) as in Fig. 1. OCRs are normalized to the third baseline measurement prior to the addition of glutamate or control. Representative traces are mean ± SD from three wells. <b>(D)</b> Mean relative respiratory capacities for the experiment depicted in (C). OCRs are normalized to the control value (black circles) corresponding to the measurement just prior to antimycin A addition in the representative time course shown in (C). Results are mean ± SE from 3 independent experiments.</p

Mg²⁺ pre-treatment or preventing NMDA receptor-mediated calcium entry improves relative respiratory capacity of Type I neurons.

<p><b>(A)</b> and <b>(B)</b> Type I neurons were pre-incubated for 1 hr in the absence or presence of MgSO₄ (3 mM), MK801 (10 µM), or the two combined prior to measurement of OCRs. Control (con), FCCP+MK801+CNQX, pyruvate (pyr), and antimycin (AA) were added sequentially as in Fig. 1, although MK801 was omitted when already present via pre-incubation. Absolute OCRs are shown in (A) and normalized OCRs are shown in (B). OCRs in (B) are normalized to the third baseline measurement prior to the control injection. Representative traces are mean ± SD from three wells. (<b>(C)</b>) Mean relative respiratory capacities for the experiment depicted in (A) and (B). OCRs are normalized to the control value (black circles) corresponding to the measurement just prior to antimycin A addition in the representative time course shown in (B). Results are mean ± SE from 6 independent experiments. *p<0.05 relative to control. #p<0.05 relative to Mg²⁺ alone. (<b>(D)</b>) Mean relative respiratory capacities for Type I neurons pre-incubated for 1 hr in low Ca²⁺ aCSF containing 5 mM EGTA, or normal aCSF with or without the added presence of tetrodotoxin (TTX, 100 nM) or MK801 (10 µM). Treatments were also present throughout the OCR measurements. Drug additions were performed as in (A) and relative respiratory capacity was calculated as in (C). Results are mean ± SE from 3 independent experiments. *p<0.05 relative to control.</p

Glutamate receptor antagonists decrease basal respiration in Type I but not Type II neurons.

<p>Basal O₂ consumption rates (OCRs) were measured, and then MK801+CNQX (10 µM each, triangles) or control (con, circles) was added at the arrow to Type I or Type II neurons (closed and open symbols, respectively). Results are mean ± SD from 3 wells.</p

MgSO₄ pre-treatment abolishes excitotoxic glutamate-induced ATP depletion.

<p>Neurons were pre-incubated for 1 hr in the absence or presence of 5 µM glutamate (glu) with the added absence or presence of 3 mM MgSO₄. Neurons were then incubated for an additional 2 hr with 100 µM glu or control and total cellular ATP was measured. Results are mean ± SE from 3 independent experiments performed in triplicate and are normalized to total protein. *p<0.05 relative to control.</p