TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice

Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base 1 (Sptlc1) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc11 and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc1^+/?, Sptlc2^+/?) are healthy. Analysis showed that, compared with WT mice, Sptlc1^+/? and Sptlc2^+/? mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57% (P < 0.01 and P < 0.0001, respectively); (2) decreased liver Sptlc1 mass by 50% and Sptlc2 mass by 70% (P < 0.01 and P < 0.01, respectively), moreover, Sptlc1 mass decreased by 70% in Sptlc2^+/? mouse liver, while Sptlc2 mass decreased by 53% in Sptlc1^+/? mouse liver (P < 0.001 and P < 0.01, respectively); (3) decreased liver SPT activity by 45% and 60% (P < 0.01, respectively); (4) decreased liver ceramide (22% and 39%, P < 0.05 and P < 0.01, respectively) and sphingosine levels (22% and 31%, P < 0.05 and P < 0.01, respectively); (5) decreased plasma ceramide (45% and 39%, P < 0.01, respectively), sphingosine-1-phosphate (31% and 32%, P < 0.01, respectively) and sphingosine levels (22.5% and 25%, P < 0.01, respectively); (6) dramatically decreased plasma lysosphingomyelin (17-fold and 16-fold, P < 0.0001, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc1 and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the course of such diseases as atherosclerosis.     

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2 + Entry

Store-operated Ca^2 + entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca^2 + entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca^2 + release-activated Ca^2 + channel current (I_CRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and I_CRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca²⁺ depletion-dependent oligomerization of the luminal Ca^2 +-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.     

Brain-Derived Neurotrophic Factor Prevents Depressive-Like Behaviors in Early-Symptomatic YAC128 Huntington’s Disease Mice

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the Huntington disease gene. The symptomatic stage of the disease is defined by the onset of motor symptoms. However, psychiatric disturbances, including depression, are common features of HD and can occur a decade before the manifestation of motor symptoms. We used the YAC128 transgenic mice (which develop motor deficits at a later stage, allowing more time to study depressive behaviors without the confounding effects of motor impairment) to test the effects of intranasal brain-derived neurotrophic factor (BDNF) treatment for 15 days in the occurrence of depressive-like behaviors. Using multiple well-validated behavioral tests, we found that BDNF treatment alleviated anhedonic and depressive-like behaviors in the YAC128 HD mice. Furthermore, we also investigated whether the antidepressant-like effects of BDNF were associated with an increase in adult hippocampal neurogenesis. However, BDNF treatment only increased cell proliferation and neuronal differentiation in the hippocampal dentate gyrus (DG) of wild-type (WT) mice, without altering these parameters in their YAC128 counterparts. Moreover, BDNF treatment did not cause an increase in the number of dendritic branches in the hippocampal DG when compared with animals treated with vehicle. In conclusion, our results suggest that non-invasive administration of BDNF via the intranasal route may have important therapeutic potential for treating mood disturbances in early-symptomatic HD patients.     

Bone-marrow cell therapy induces differentiation of radial glia-like cells and rescues the number of oligodendrocyte progenitors in the subventricular zone after global cerebral ischemia

The subventricular zone (SVZ) is recognized as one of the neurogenic regions in the adult mammalian central nervous system and the presence of cells that share similar characteristics with developmental radial glia, the radial glia-like cells (RGLCs) has been demonstrated in this region. In this study, we investigated whether and how SVZ cells respond to global ischemia and/or to the intravenous transplant of bone-marrow mononuclear cells (BMMCs). Adult rats were subjected to bilateral common carotid ligation (BCCL) and after 1 day 2 × 10⁷ BMMCs or saline injection. The BMMC transplant stimulated a transitory increase in the proliferation of SVZ cells in the BCCL group. We observed a significant increase in the number of RGLCs 3 days after ischemia, in both BCCL and BCCL + BMMC groups. However, this increase persisted in the subsequent days only in BCCL animals that received the transplant. BMMC transplantation also inhibits the reduction of NG2-positive oligodendrocyte progenitors in the SVZ observed in the BCCL group. Interestingly, brain-derived neurotrophic factor (BDNF) expression was up-regulated in the SVZ in the treated animals, but not in the other groups. These data thus suggest that BMMC transplantation modulates the phenotype of RGLCs/progenitors in the SVZ and could have a protective role after ischemia.     

Pulsed electromagnetic field enhances brain-derived neurotrophic factor expression through L-type voltage-gated calcium channel- and Erk-dependent signaling pathways in neonatal rat dorsal root ganglion neurons

Although pulsed electromagnetic field (PEMF) exposure has been reported to promote neuronal differentiation, the mechanism is still unclear. Here, we aimed to examine the effects of PEMF exposure on brain-derived neurotrophic factor (Bdnf) mRNA expression and the correlation between the intracellular free calcium concentration ([Ca²⁺]_i) and Bdnf mRNA expression in cultured dorsal root ganglion neurons (DRGNs). Exposure to 50 Hz and 1 mT PEMF for 2 h increased the level of [Ca²⁺]_i and Bdnf mRNA expression, which was found to be mediated by increased [Ca²⁺]_i from Ca²⁺ influx through L-type voltage-gated calcium channels (VGCCs). However, calcium mobilization was not involved in the increased [Ca²⁺]_i and BDNF expression, indicating that calcium influx was one of the key factors responding to PEMF exposure. Moreover, PD098059, an extracellular signal-regulated kinase (Erk) inhibitor, strongly inhibited PEMF-dependant Erk1/2 activation and BDNF expression, indicating that Erk activation is required for PEMF-induced upregulation of BDNF expression. These findings indicated that PEMF exposure increased BDNF expression in DRGNs by activating Ca²⁺- and Erk-dependent signaling pathways.     

Baclofen,a GABAB receptor agonist,enhances ubiquitin-proteasome system functioning and neuronal survival in Huntington’s disease model mice

Huntington’s disease (HD) is an autosomal neurodegenerative disease. Its manifestations is selective degeneration of medium-sized spiny neurons (MSN) in the striatum. The specificity of the vulnerability of these GABAergic MSNs can be explained by abnormal protein accumulation, excitotoxicity, mitochondrial dysfunction, and failure of trophic control, among other dysfunctions. In this study, we used in vitro and in vivo models of HD to study the effects of GABAergic neuron stimulation on the cellular protein degradation machinery. We administered the GABA_B receptor agonist, baclofen, to wild-type or mutant huntingtin-expressing striatal cells (HD19 or HD43). Chymotrypsin-like proteasome activity and cell viability were significantly increased in the mutant huntingtin-expressing striatal cells (HD43) after GABA_B receptor agonist treatment. In addition, we systemically administered baclofen to a HD model containing the entire human huntingtin gene with 128 CAG repeats (YAC128). Chymotrypsin-like proteasome activity was significantly increased in YAC128 transgenic mice after baclofen administration. Baclofen-injected mutant YAC128 mice also showed significantly reduced numbers of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum. Baclofen markedly improved behavioral abnormalities in mutant YAC128 mice as determined by the rotarod performance test. These data indicate that stimulation of GABAergic neurons with the GABA_B receptor agonist, baclofen, enhances ubiquitin-proteasome system (UPS) function and cell survival in in vitro and in vivo models of HD.     

Factors influencing diurnal variation in height among adults

This study evaluates height loss during the day in light of variables assessable through participant self-report. Participants were 19 female and 15 male staff and students who met with us twice with a mean interval between measurement sessions of 6.96 h (SD = 0.86). Hypotheses were evaluated using two-stage least squares regression analysis (SYSTAT 10). Males and females lost similar amounts of height between sessions $(\text{male},\overline{x}=6.9\text{mm};\text{female},\overline{x}=7.4\text{mm})$. Among factors considered to impact overall height loss, sleep duration (Adj. R² = 0.181, p = 0.022) and height (Adj. R² = 0.121, p = 0.048) were useful in univariate analyses, though they were not significant in any models that included variables other than sex. Judging from log-transformed BMI variation, heavier males and females lost more height (F_{(2, 31)} = 4.59, Adj. R² = 0.179, p = 0.018). Among factors anticipated to reduce height loss by acting prior to morning measurements, only time spent walking was significantly associated (β = 2.6 ± 0.8 mm, t = 3.16, p = 0.004) when included as a predictor along with sex (p = 0.17) and log-BMI (p = 0.003). This model explained about 38% of height loss variance. None of the factors considered as potentially acting between measurement sessions showed statistically significant influences when included in the model just described, though predictor coefficients were in the anticipated direction. Results suggest that self-reported activities may be an important supplement to anthropometric studies, both for planning and later evaluation, particularly in large studies.     

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’

We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus ‘Candidatus Brocadia’ with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized ‘Ca. Brocadia fulgida’ and ‘Ca. Brocadia sinica’ with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20–45 °C with a maximum activity at 37 °C. The maximum specific growth rate (μ_max) was 0.0082 h^?1 at 37 °C, corresponding to a doubling time of 3.5 days. The half-saturation constant (K_S) for nitrite was 5 ± 2.5 μM. The anammox activity was inhibited by nitrite (IC₅₀ = 11.6 mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with ‘Ca. Brocadia sinica’ and ‘Ca. Brocadia fulgida’. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name ‘Ca. Brocadia sapporoensis’ sp. nov.     

The mitochondrial calcium uniporter is involved in mitochondrial calcium cycle dysfunction: Underlying mechanism of hypertension associated with mitochondrial tRNAIle A4263G mutation

Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNA^Ile A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca²⁺ cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNA^Ile A4263G mutation. The mitochondrial calcium ([Ca²⁺]_m) in cells from hypertensive subjects with the tRNA^Ile A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P < 0.05). Meanwhile, cytosolic calcium ([Ca²⁺]_c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca²⁺]_c by activating ryanodine receptor on endoplasmic reticulum, [Ca²⁺]_c/[Ca²⁺]_m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P < 0.05). [Ca²⁺]_c increased and [Ca²⁺]_m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca²⁺ uptake into the mitochondria, and cytoplasmic Ca²⁺ overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNA^Ile A4263G mutation.     

Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

Background

Dimebon is an antihistamine compound with a long history of clinical use in Russia. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders. It has demonstrated efficacy in phase II Alzheimer's disease (AD) and Huntington's disease (HD) clinical trials. The mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain unclear. It has been suggested that Dimebon may act by blocking NMDA receptors or voltage-gated Ca²⁺ channels and by preventing mitochondrial permeability pore transition.

Results

We evaluated the effects of Dimebon in experiments with primary striatal neuronal cultures (MSN) from wild type (WT) mice and YAC128 HD transgenic mice. We found that Dimebon acts as an inhibitor of NMDA receptors (IC50 = 10 μM) and voltage-gated calcium channels (IC50 = 50 μM) in WT and YAC128 MSN. We further found that application of 50 μM Dimebon stabilized glutamate-induced Ca²⁺ signals in YAC128 MSN and protected cultured YAC128 MSN from glutamate-induced apoptosis. Lower concentrations of Dimebon (5 μM and 10 μM) did not stabilize glutamate-induced Ca²⁺ signals and did not exert neuroprotective effects in experiments with YAC128 MSN. Evaluation of Dimebon against a set of biochemical targets indicated that Dimebon inhibits α-Adrenergic receptors (α_1A, α_1B, α_1D, and α_2A), Histamine H₁ and H₂ receptors and Serotonin 5-HT_2c, 5-HT_5A, 5-HT₆ receptors with high affinity. Dimebon also had significant effect on a number of additional receptors.

Conclusion

Our results suggest that Ca²⁺ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca²⁺ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.     

Endogenous osteopontin induces myocardial CCL5 and MMP-2 activation that contributes to inflammation and cardiac remodeling in a mouse model of chronic Chagas heart disease

Cardiac dysfunction with progressive inflammation and fibrosis is a hallmark of Chagas disease caused by persistent Trypanosoma cruzi infection. Osteopontin (OPN) is a pro-inflammatory cytokine that orchestrates mechanisms controlling cell recruitment and cardiac architecture. Our main goal was to study the role of endogenous OPN as a modulator of myocardial CCL5 chemokine and MMP-2 metalloproteinase, and its pathological impact in a murine model of Chagas heart disease. Wild-type (WT) and OPN-deficient (spp1 ?/?) mice were parasite-infected (Brazil strain) for 100 days. Both groups developed chronic myocarditis with similar parasite burden and survival rates. However, spp1 ?/? infection showed lower heart-to-body ratio (P < 0.01) as well as reduced inflammatory pathology (P < 0.05), CCL5 expression (P < 0.05), myocyte size (P < 0.05) and fibrosis (P < 0.01) in cardiac tissues. Intense OPN labeling was observed in inflammatory cells recruited to infected heart (P < 0.05). Plasma concentration of MMP-2 was higher (P < 0.05) in infected WT than in spp1 ?/? mice. Coincidently, specific immunostaining revealed increased gelatinase expression (P < 0.01) and activity (P < 0.05) in the inflamed hearts from T. cruzi WT mice, but not in their spp1 ?/? littermates. CCL5 and MMP-2 induction occurred preferentially (P < 0.01) in WT heart-invading CD8⁺ T cells and was mediated via phospho-JNK MAPK signaling. Heart levels of OPN, CCL5 and MMP-2 correlated (P < 0.01) with collagen accumulation in the infected WT group only. Endogenous OPN emerges as a key player in the pathogenesis of chronic Chagas heart disease, through the upregulation of myocardial CCL5/MMP-2 expression and activities resulting in pro-inflammatory and pro-hypertrophic events, cardiac remodeling and interstitial fibrosis.     

68Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors

Molecular imaging using radiolabeled Tyrosine Kinase Inhibitors (TKI) is a promising strategy for detection and staging of EGFR-positive cancers. A novel analogue of one such TKI, Erlotinib has been developed for PET imaging by derivatizing the parent Erlotinib molecule for conjugation with the bifunctional chelator p-SCN-Bn-NOTA towards radiolabeling with ⁶⁸Ga. NOTA-Erlotinib conjugate was synthesized and characterized by NMR and ESI-MS techniques. The conjugate was radiolabeled with ⁶⁸Ga in 95 ± 2% yield, as evidenced by HPLC characterization. The log P value of ⁶⁸Ga-NOTA-Erlotinib was – (0.6 ± 0.1). The ⁶⁸Ga-NOTA-Erlotinib conjugate was characterized using its ^natGa-NOTA-Erlotinib surrogate. Cell viability studies showed that the NOTA-Erlotinib conjugate retained the biological efficacy of the parent Erlotinib molecule. Further, ⁶⁸Ga-NOTA-Erlotinib exhibited an uptake of 9.8 ± 0.4% in A431 cells which was inhibited by 55.1 ± 0.2% on addition of cold Erlotinib (10 µg) confirming the specificity of the radioconjugate for EGFR expressing cells. In the biodistribution studies carried out in tumor bearing SCID mice, ⁶⁸Ga-NOTA-Erlotinib conjugate showed moderate tumor accumulation (1.5 ± 0.1% ID/g at 30 min p.i.; 0.7 ± 0.2% ID/g at 1 h p.i.). Hepatobiliary clearance of the radioconjugate was observed. The ⁶⁸Ga-NOTA-Erlotinib conjugate was found to have high in vivo stability as determined by the metabolite analysis study using urine sample of the Swiss mice injected with the preparation. The overall properties of ⁶⁸Ga-NOTA-Erlotinib are promising and merit further exploration. To the best of our knowledge, this is the first report on the design of a ⁶⁸Ga labeled Erlotinib for PET imaging of EGFR and opens avenues for the successful development of ⁶⁸Ga labeled TKI for imaging of EGFR over-expressing tumors.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Ca2+ handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease

We investigated Ca²⁺ handling in isolated brain synaptic and non‐synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non‐synaptic mitochondria from 2‐ and 12‐month‐old YAC128 mice had larger Ca²⁺ uptake capacity than mitochondria from YAC18 and wild‐type FVB/NJ mice. Synaptic mitochondria from 12‐month‐old YAC128 mice had further augmented Ca²⁺ capacity compared with mitochondria from 2‐month‐old YAC128 mice and age‐matched YAC18 and FVB/NJ mice. This increase in Ca²⁺ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12‐month‐old YAC128 mice. We speculate that this may happen because of mHtt‐mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca²⁺‐induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca²⁺ followed by recovery to near resting levels. Following recovery of cytosolic Ca²⁺, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca²⁺, suggesting similar Ca²⁺ release and, consequently, Ca²⁺ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca²⁺ handling in brain mitochondria of YAC128 mice.

     

‘Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. We describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L^?1 d^?1. The DCM degrader attained 5.25 × 10⁷ ± 1.0 × 10⁷ cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.     

Anthropometric dimensions provide reliable estimates of abdominal adiposity: A validation study

Abdominal fat accumulation is a major risk factor for cardiometabolic morbidity and mortality. The purpose of the study is to assess the possibility of developing accurate estimation equations based on body measurements to determine total abdominal (TFA), subcutaneous (SFA) and visceral fat area (VFA). Hungarian volunteers (n = 198) aged between 20 and 81 years were enrolled in the study, which was conducted between July and November 2014. All persons underwent anthropometric measurements and computer tomographic (CT) scanning. Sex-specific multiple linear regression analyses were conducted in a subgroup of 98 participants to generate estimation models, then Bland–Altman's analyses were applied in the cross-validation group to compare their predictive efficiency. The variables best predicting VFA were hip circumference, calf circumference and waist-to-hip ratio (WHR) for males (R² = 0.713; SEE = 5602.1 mm²) and sagittal abdominal diameter (SAD), WHR, thigh circumference and triceps skinfold for females (R² = 0.845; SEE = 3835.6 mm²). The SFA prediction equation included SAD, thigh circumference and abdominal skinfold for males (R² = 0.848; SEE = 4124.1 mm²), body mass index and thigh circumference for females (R² = 0.861; SEE = 5049.7 mm²). Prediction accuracy was the highest in the case of TFA: hip circumference and WHR for males (R² = 0.910; SEE = 5637.2 mm²), SAD, thigh circumference and abdominal skinfold for females (R² = 0.915; SEE = 6197.5 mm²) were used in the equations. The results suggested that deviations in the predictions were independent of the amount of adipose tissue. Estimation of abdominal fat depots based on anthropometric traits could provide a cheap, reliable method in epidemiologic research and public health screening to evaluate the risk of cardiometabolic events.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.