Two potential equilibrium states in long-term soil respiration activity of dry grasslands are maintained by local topographic features.

Soil respiration of grasslands is spatio-temporally variable reflecting the changing biological activities of the soil. In our study we analysed how the long-term soil respiration activities of dry grasslands would perform in terms of resistance and resilience. We also investigated how terrain features are responsible for response stability. We conducted a 7-year-long spatial study in a Hungarian dry grassland, measuring soil respiration (Rs), soil temperature (Ts) and soil water content (SWC) along 15 measuring campaigns in 80 × 60 m grids and soil organic carbon content in 6 of the occasions. Two proxy variables were introduced to grasp the overall Rs activity, as well as its temporal stability: average rankRs, the temporal average Rs rank of a measuring position from the campaigns revealed the persistent spatial pattern of Rs, while rangeRs, the range of ranks of the positions from the campaigns described the amplitude of the Rs response in time, referring to the response stability in terms of resistance or resilience. We formulated a hypothetic concept of a two-state equilibrium to describe the performance of the long-term Rs activity: Rs activity with smaller rangeRs, that is both the lower elevation positions with larger rankRs ("state I") and the higher elevation positions with smaller rankRs ("state II") correspond to an equilibrium state with several terrain attributes being responsible for the equilibrium responses. Majority of the measuring positions was belonging to none of these equilibrium states. These positions showed higher rangeRs for medium rankRs, suggesting resilience (not resistance) as a major strategy for this ecosystem.

Automated material map generation from MRI scout pairs for preclinical PET attenuation correction.

A novel method is presented to perform material map segmentation from preclinical MRI for corresponding PET attenuation correction. MRI does not provide attenuation ratio, hence segmenting a material map from it is challenging. Furthermore the MRI images often suffer from ghost artifacts. On the contrary MRI has no radiation dose. Our method operated with fast spin echo scout pairs that had perpendicular frequency directions. This way the direction of the ghost artifacts were perpendicular as well. Our body-air segmentation method built on this a priori information and successfully erased the ghost artifacts from the final binary mask. Visual and quantitative validation was performed by two preclinical specialists. Results indicate that our method is effective against MRI scout ghost artifacts and that PET attenuation correction based on MRI makes sense even on preclinical images.

Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of NRF2-mediated antioxidant response.

There is strong evidence showing that aging is associated with vascular oxidative stress, which has been causally linked to the development of cardiovascular diseases. NF-E2-related factor-2 (Nrf2) is a transcription factor, which is activated by reactive oxygen species in the vasculature of young animals leading to the upregulation of various antioxidant genes. The present study was designed to elucidate age-related changes in the homeostatic role of Nrf2-driven free radical detoxification mechanisms in the vasculature. We found that in the aorta of Fischer 344 × Brown Norway rats, aging results in a progressive increase in O(2)(·-) production, and downregulates protein and mRNA expression of Nrf2, which is associated with a decreased nuclear Nrf2 activity and a decrease in the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1, γ-glutamylcysteine synthetase, and heme oxygenase-1. There was an inverse relationship between vascular expression of Nrf2 target genes and age-related increases in the expression of the NF-κB target genes ICAM-1 and IL-6, which was significant by regression analysis. In cultured aorta segments of young (3 mo old) rats treatment with H(2)O(2) and high glucose significantly increases nuclear translocation of Nrf2 and upregulates the expression of Nrf2 target genes. In contrast, in cultured aorta segments of aged (24 mo old) rats, the induction of Nrf2-dependent responses by H(2)O(2) and high glucose are blunted. High glucose-induced vascular oxidative stress was more severe in aortas of aged rats, as shown by the significantly increased H(2)O(2) production in these vessels, compared with responses obtained in aortas from young rats. Moreover, we found that aging progressively increases vascular sensitivity to the proapoptotic effects of H(2)O(2) and high glucose treatments. Taken together, aging is associated with Nrf2 dysfunction in the vasculature, which likely exacerbates age-related cellular oxidative stress and increases sensitivity of aged vessels to oxidative stress-induced cellular damage.

Free radical production, antioxidant capacity, and oxidative stress response signatures in fibroblasts from Lewis dwarf rats: effects of life span-extending peripubertal GH treatment.

The discovery that in invertebrates, disruption of the insulin/insulin-like growth factor (IGF)-1 pathway extends life span and increases resistance to oxidative injury led to the hypothesis that IGF-1 signaling may play a role in regulating cellular reactive oxygen species production, oxidative stress resistance, and consequentially, organismal life span in mammals. However, previous studies testing this hypothesis in rodent models of IGF-1 deficiency yielded controversial results. The Lewis dwarf rat is a useful model of human growth hormone (GH)/IGF-1 deficiency as it mimics many of the pathophysiological alterations present in human GH/IGF-1-deficient patients as well as elderly individuals. Peripubertal treatment of Lewis dwarf rats with GH results in a significant extension of life span. The present study was designed to test the role of the GH/IGF-1 axis in regulating cellular oxidative stress and oxidative stress resistance, utilizing primary fibroblasts derived from control rats, Lewis dwarf rats and GH-replete dwarf rats. Measurements of cellular dihydroethidium and C-H(2)DCFDA fluorescence showed that cellular O(2)(·-) and peroxide production were similar in each group. Fibroblasts from control and Lewis dwarf rats exhibited similar antioxidant capacities and comparable sensitivity to H(2)O(2), rotenone, high glucose, tunicamycin, thapsigargin, paraquat, and mitomycin, which cause apoptosis through increasing oxidative stress, mitochondrial damage, ATP depletion, and/or by damaging DNA, lipids and proteins. Fibroblasts from GH-replete rats exhibited significantly increased antioxidant capacities and superior resistance to H(2)O(2), rotenone and bacterial lipopolysaccharide-induced cell death compared with cells from Lewis dwarf rats, whereas their sensitivity to the other stressors investigated was not statistically different. Thus, low circulating IGF-1 levels present in vivo in Lewis dwarf rats do not elicit long-lasting alterations in cellular reactive oxygen species generation and oxidative stress resistance, whereas life span-extending peripubertal GH treatment resulted in increased antioxidant capacity and increased resistance to cellular injury caused by some, but not all, oxidative stressors.

Vasoprotective effects of life span-extending peripubertal GH replacement in Lewis dwarf rats.

In humans, growth hormone deficiency (GHD) and low circulating levels of insulin-like growth factor 1 (IGF-1) significantly increase the risk for cerebrovascular disease. Genetic growth hormone (GH)/IGF-1 deficiency in Lewis dwarf rats significantly increases the incidence of late-life strokes, similar to the effects of GHD in elderly humans. Peripubertal treatment of Lewis dwarf rats with GH delays the occurrence of late-life stroke, which results in a significant extension of life span. The present study was designed to characterize the vascular effects of life span-extending peripubertal GH replacement in Lewis dwarf rats. Here, we report, based on measurements of dihydroethidium fluorescence, tissue isoprostane, GSH, and ascorbate content, that peripubertal GH/IGF-1 deficiency in Lewis dwarf rats increases vascular oxidative stress, which is prevented by GH replacement. Peripubertal GHD did not alter superoxide dismutase or catalase activities in the aorta nor the expression of Cu-Zn-SOD, Mn-SOD, and catalase in the cerebral arteries of dwarf rats. In contrast, cerebrovascular expression of glutathione peroxidase 1 was significantly decreased in dwarf vessels, and this effect was reversed by GH treatment. Peripubertal GHD significantly decreases expression of the Nrf2 target genes NQO1 and GCLC in the cerebral arteries, whereas it does not affect expression and activity of endothelial nitric oxide synthase and vascular expression of IGF-1, IGF-binding proteins, and inflammatory markers (tumor necrosis factor alpha, interluekin-6, interluekin-1ß, inducible nitric oxide synthase, intercellular adhesion molecule 1, and monocyte chemotactic protein-1). In conclusion, peripubertal GH/IGF-1 deficiency confers pro-oxidative cellular effects, which likely promote an adverse functional and structural phenotype in the vasculature, and results in accelerated vascular impairments later in life.

Mitochondrial Ca2+ uptake is inhibited by a concerted action of p38 MAPK and protein kinase D.

Angiotensin II elicits cytosolic Ca2+ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake, Cell Calcium 43 (2008) 250-259]. Now we report that simultaneous activation of these protein kinases (by TNFalpha and PMA+an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca2+ signal, elicited by depolarisation or store-operated Ca2+ influx, into the mitochondria. The Ca2+ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca2+] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca2+ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca2+ signal from the cytosol into the mitochondria. Our data indicate that Ca2+ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca2+ uptake, thus reducing the risk of Ca2+ overload.

Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake.

Angiotensin II elicits cytosolic and mitochondrial Ca2+ signal in H295R adrenocortical cells. We found that Ca2+ uptake rate and peak values in small mitochondrial regions both depend on the colocalization of these mitochondrial regions with GFP-marked endoplasmic reticular (ER) vesicles. The dependence of the Ca2+ response on this colocalization is abolished by SB202190 and PD169316, inhibitors of p38 MAPK, as well as by transfection with siRNA against p38 MAPK mRNA. The same manoeuvres result in an increased ratio of global mitochondrial to global cytosolic Ca2+ response, indicating that inhibition of p38 MAPK is followed by enhanced mitochondrial Ca2+ uptake. alpha-Toxin and TNFalpha, agents which similarly to angiotensin II increase the phosphorylation of p38, failed to affect mitochondrial Ca2+ uptake, indicating that activation of p38 MAPK is necessary but not sufficient for the inhibition of Ca2+ uptake. Bisindolylmaleimide, an inhibitor of the conventional and novel-type protein kinase C isoforms also evokes enhanced mitochondrial Ca2+ uptake, whereas Gö6976 that inhibits the conventional isoforms only failed to exert any effect. These data show that angiotensin II attenuates Ca2+ uptake predominantly into mitochondria that do not colocalize with ER, by a mechanism involving p38 MAPK and a novel-type PKC.

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains.

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.

Reactive oxygen species, Ca2+ signaling and mitochondrial NAD(P)H level in adrenal glomerulosa cells.

The acute effects of ultraviolet light, the superoxide-generating xanthine-xanthine oxidase system and H(2)O(2) to on calcium signaling and mitochondrial pyridine nucleotide metabolism were investigated in rat glomerulosa cells. UV light induced the formation of superoxide, that, similar to exogenously applied superoxide and H(2)O(2), decreased the level of mitochondrial NAD(P)H. Free radical scavengers antagonized this effect of UV light. Extracellularly generated superoxide elicited Ca(2+) transients and inhibited angiotensin II-induced cytoplasmic Ca(2+) signaling. Low intensity UV light did not affect basal [Ca(2+)] and failed to influence Ca(2+) signaling induced by depolarization or store depletion. UV light of the same low power reduced both cytoplasmic and mitochondrial Ca(2+) signals induced by angiotensin II. The lack of UV effect on inositol phosphate formation indicates that the inhibition of cytoplasmic Ca(2+) signaling is due to reduced Ca(2+) release from InsP(3)-sensitive stores. Decreased mitochondrial Ca(2+) uptake may be attributed to UV-induced perturbation of the perimitochondrial microdomain.

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells.

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.