ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure.

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering.

Cellular energy required for the maintenance of cellular life is stored in the form of adenosine triphosphate (ATP). Understanding cellular mechanisms, including ATP-dependent metabolisms, is crucial for disease diagnosis and treatment, including drug development and investigation of new therapeutic systems. As an ATP-dependent metabolism, endocytosis plays a key role not only in the internalization of molecules but also in processes including cell growth, differentiation, and signaling. To understand cellular mechanisms including endocytosis, many techniques ranging from molecular approaches to spectroscopy are used. Surface-enhanced Raman scattering (SERS) is shown to provide valuable label-free molecular information from living cells. In this study, receptor-mediated endocytosis was investigated with SERS by inhibiting endocytosis with ATP depletion agents: sodium azide (NaN3) and 2-deoxy-d-glucose (dG). Human lung bronchial epithelium (Beas-2b) cells, normal prostate epithelium (PNT1A) cells, and cervical cancer epithelium (HeLa) cells were used as models. First, the effect of NaN3 and dG on the cells were examined through cytotoxicity, apoptosis-necrosis, ATP assay, and uptake inhibition analysis. An attempt to relate the spectral changes in the cellular spectra to the studied cellular events, receptor-mediated endocytosis inhibition, was made. It was found that the effect of two different ATP depletion agents can be discriminated by SERS, and hence receptor-mediated endocytosis can be tracked from single living cells with the technique without using a label and with limited sample preparation.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

Iron is a ligand of SecA-like metal-binding domains in vivo.

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.

Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.

Effect of Sodium Azide on Quantitative and Qualitative Stem Traits in the M2 Generation of Ethiopian Sesame (Sesamum indicum L.) Genotypes.

The emerging oilseed crop Sesamum indicum, also known as the queen of oilseeds, is being grown globally for its oil content for medicinal and nutritional values. One of the key challenges of sesame cultivation is its low productivity. In the present study, sodium azide (NaN3) was used as a chemical mutagen. The aim of this study was to examine the effect of NaN3 on quantitative and qualitative stem traits in the M2 generation of Ethiopian sesame (Sesamum indicum L.) genotypes. Seeds of fourteen sesame genotypes were used in this study and germinated and grown under greenhouse conditions. Different qualitative and quantitative data were collected and analyzed. Traits such as plant height, ground distance to first distance, and internode length were significantly affected by NaN3 treatment. The highest plant height was recorded in the control on Humera 1 and Baha Necho genotypes, while the lowest was observed on Setit 2 and Hirhir treated with the chemical. The highest ground distance to the first branch was observed in Gumero, while the least ground distance was recorded in Setit 1 in the treated and control genotypes, respectively. The best internode length was recorded on Setit 2 and ADI in the control, while the lowest internode length was observed in Setit 1 genotype treated with sodium azide. Genotypes such as ACC44, ADI, Baha Necho, Borkena, Gonder 1, and Setit 1 treated with NaN3 have showed glabrous type of stem hairiness. All the fourteen genotypes (both treated and control) were clustered into four groups. In conclusion, we observed a highly significant variation among the genotypes due the effect of the chemical and genotypes themselves. Hence, this report would create more genetic diversity for further sesame genetic research improvements.

Novel polymeric microspheres: Synthesis, enzyme immobilization, antimutagenic activity, and antimicrobial evaluation against pathogenic microorganisms.

New polymeric microspheres containing azomethine (1a-1c and 2a-2c) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT-IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis-Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (Vmax ) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well-diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.

Cisplatin-Induced Giant Cells Formation Is Involved in Chemoresistance of Melanoma Cells.

Melanoma is notoriously resistant to current cancer therapy. However, the chemoresistance mechanism of melanoma remains unclear. The present study unveiled that chemotherapy drug cisplatin induced the formation of giant cells, which exhibited enlargement in cell diameter and nucleus in mice and human melanoma cells. Giant cells were positive with melanoma maker S100 and cancer stem cell markers including ABCB5 and CD133 in vitro and in vivo. Moreover, giant cells retained the mitotic ability with expression of proliferation marker Ki-67 and exhibited multiple drug resistance to doxorubicin and actinomycin D. The mitochondria genesis/activities and cellular ATP level were significantly elevated in giant cells, implicating the demand for energy supply. Application of metabolic blockers such as sodium azide or 2-deoxy glucose abolished the cisplatin-induced giant cells formation and expression of cancer stemness markers. The present study unveils a novel chemoresistance mechanism of melanoma cells via size alteration and the anti-neoplastic strategy by targeting giant cells.

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.

Neuroprotective Role of Selected Antioxidant Agents in Preventing Cisplatin-Induced Damage of Human Neurons In Vitro.

Chemotherapy-induced peripheral neuropathy (CIPN) is a side effect of platinum-based chemotherapy and decreases the quality of life of cancer patients. We compared neuroprotective properties of several agents using an in vitro model of terminally differentiated human cells NT2-N derived from cell line NT2/D1. Sodium azide and an active metabolite of amifostine (WR1065) increase cell viability in simultaneous treatment with cisplatin. In addition, WR1065 protects the non-dividing neurons by decreasing cisplatin caused oxidative stress and apoptosis. Accumulation of Pt in cisplatin-treated cells was heterogeneous, but the frequency and concentration of Pt in cells were lowered in the presence of WR1065 as shown by X-ray fluorescence microscopy (XFM). Transition metals accumulation accompanied Pt increase in cells; this effect was equally diminished in the presence of WR1065. To analyze possible chemical modulation of Pt-DNA bonds, we examined the platinum LIII near edge spectrum by X-ray absorption spectroscopy. The spectrum found in cisplatin-DNA samples is altered differently by the addition of either WR1065 or sodium azide. Importantly, a similar change in Pt edge spectra was noted in cells treated with cisplatin and WR1065. Therefore, amifostine should be reconsidered as a candidate for treatments that reduce or prevent CIPN.

The Differential Expression of Mitochondrial Function-Associated Proteins and Antioxidant Enzymes during Bovine Herpesvirus 1 Infection: A Potential Mechanism for Virus Infection-Induced Oxidative Mitochondrial Dysfunction.

Reactive oxidative species (ROS) are important inflammatory mediators. Electrons escaping from the mitochondrial electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) in the mitochondrial respiratory chain (RC) complexes contribute to ROS production. The cellular antioxidant enzymes are important for maintaining ROS release at the physiological levels. It has been reported that BoHV-1 infection induces overproduction of ROS and oxidative mitochondrial dysfunction in cell cultures. In this study, we found that chemical interruption of RC complexes by TTFA (an inhibitor of RC complex II), NaN3 (an inhibitor of RC complex IV), and oligomycin A (an inhibitor of ATP synthase) consistently decreased virus productive infection, suggesting that the integral processes of RC complexes are important for the virus replication. The virus infection significantly increased the expression of subunit SDHB (succinate dehydrogenase) and MTCO1 (cytochrome c oxidase subunit I), critical components of RC complexes II and IV, respectively. The expression of antioxidant enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase (CAT), and glutathione peroxidase 4 (GPX4) was differentially affected following the virus infection. The protein TFAM (transcription factor A, mitochondrial) stimulated by either nuclear respiratory factor 1 (NRF1) or NRF2 is a key regulator of mitochondrial biogenesis. Interestingly, the virus infection at the late stage (at 16 h after infection) stimulated TFAM expression but decreased the levels of both NRF1 and NRF2, indicating that virus infection activated TFAM signaling independent of either NRF1 or NRF2. Overall, this study provided evidence that BoHV-1 infection altered the expression of molecules associated with RC complexes, antioxidant enzymes, and mitochondrial biogenesis-related signaling NRF1/NRF2/TFAM, which correlated with the previous report that virus infection induces ROS overproduction and mitochondrial dysfunction.

Respiratory analysis as a tool to detect physiological changes in Anisakis larvae subjected to stress.

Human infection due to eating fish parasitized by live Anisakis larvae in the third stage is considered an important health problem, and the application of treatments to ensure their mortality in the fish products is crucial to prevent the risk of infection. Mobility is used to assess viability, but mobile larvae may not always be infective and immobile larvae may be erroneously considered as non-viable. The objective was to establish whether the analysis of respiratory activity by means of the oxygen consumption rate (OCR) of Anisakis could be used to identify subtle differences between larvae that were still considered viable in terms of their mobility but had been subjected to thermal and/or chemical stress. The metabolic modulators FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone] and sodium azide were used and the basal, maximum, spare and residual respiration rates calculated. Results showed that maximum respiratory capacity of larvae subjected to freezing significantly decreased immediately after thawing, but after some acclimatization, they recovered their capacity fully. However, when these larvae were stored at 4.6 °C, their mitochondria became dysfunctional faster than those of untreated larvae. OCR also showed that mitochondria of larvae were affected by incubation at 37 °C in NaCl or gastric juice. To conclude, OCR of Anisakis in the presence of metabolic modulators can help to identify subtle changes that occur in the larva. These measurements could be used to characterize larvae subjected to various stresses so that a broader picture of Anisakis pathogenic potential can be gained.

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Microglial activation and the nitric oxide/cGMP/PKG pathway underlie enhanced neuronal vulnerability to mitochondrial dysfunction in experimental multiple sclerosis.

During multiple sclerosis (MS), a close link has been demonstrated to occur between inflammation and neuro-axonal degeneration, leading to the hypothesis that immune mechanisms may promote neurodegeneration, leading to irreversible disease progression. Energy deficits and inflammation-driven mitochondrial dysfunction seem to be involved in this process. In this work we investigated, by the use of striatal electrophysiological field-potential recordings, if the inflammatory process associated with experimental autoimmune encephalomyelitis (EAE) is able to influence neuronal vulnerability to the blockade of mitochondrial complex IV, a crucial component for mitochondrial activity responsible of about 90% of total cellular oxygen consumption. We showed that during the acute relapsing phase of EAE, neuronal susceptibility to mitochondrial complex IV inhibition is markedly enhanced. This detrimental effect was counteracted by the pharmacological inhibition of microglia, of nitric oxide (NO) synthesis and its intracellular pathway (involving soluble guanylyl cyclase, sGC, and protein kinase G, PKG). The obtained results suggest that mitochondrial complex IV exerts an important role in maintaining neuronal energetic homeostasis during EAE. The pathological processes associated with experimental MS, and in particular the activation of microglia and of the NO pathway, lead to an increased neuronal vulnerability to mitochondrial complex IV inhibition, representing promising pharmacological targets.

Photosensitized Protein-Damaging Activity, Cytotoxicity, and Antitumor Effects of P(V)porphyrins Using Long-Wavelength Visible Light through Electron Transfer.

Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (1O2) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of 1O2. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.

Purification and properties of Manganese Superoxide Dismutase (MnSOD) from Tamarix aphylla L.

Superoxide dismutase (SOD) of the Tamarix aphylla leaves were detected at optimum conditions that collected in April, May and June. Results indicated the specific activity in the crude extract reaching to 36.76 unit/ mg protein. Crude SOD was purified by several techniques, precipitation with ammonium sulfate (50-75) %, Ion exchange chromatography using DEAE-cellulose and two steps of size exclusion chromatography on sephacryl S-200 column. The obtained specific activity (310 unit/mg protein) and purification fold 7.91. The purified enzyme revealed one band by SDS-polyacrylamide gel electrophoresis with molecular mass 85.703 kDa. while 89.125 kDa by Sephacryl S-200. The optimal pH and temperature for enzyme activity were 7.5, and 50ºC respectively. EDTA, SDS and NaN3 reduced activity, contrariwise of H2O2 and KCN, pointed to the studied SOD is MnSOD. Michalis constant Km and maximum velocity Vmax values were 0.016 mM and 55.86 mM/min, respectively by using Pyrogallol as substrate. According to the results, we conclude Tamarix aphylla produce MnSOD which can have purified by serial purification techniques for better activity and characterized for further studies.

Comparative Proteomic Analysis during the Involvement of Nitric Oxide in Hydrogen Gas-Improved Postharvest Freshness in Cut Lilies.

Our previous studies suggested that both hydrogen gas (H2) and nitric oxide (NO) could enhance the postharvest freshness of cut flowers. However, the crosstalk of H2 and NO during that process is unknown. Here, cut lilies (Lilium "Manissa") were used to investigate the relationship between H2 and NO and to identify differentially accumulated proteins during postharvest freshness. The results revealed that 1% hydrogen-rich water (HRW) and 150 µM sodium nitroprusside (SNP) significantly extended the vase life and quality, while NO inhibitors suppressed the positive effects of HRW. Proteomics analysis found 50 differentially accumulated proteins in lilies leaves which were classified into seven functional categories. Among them, ATP synthase CF1 alpha subunit (chloroplast) (AtpA) was up-regulated by HRW and down-regulated by NO inhibitor. The expression level of LlatpA gene was consistent with the result of proteomics analysis. The positive effect of HRW and SNP on ATP synthase activity was inhibited by NO inhibitor. Meanwhile, the physiological-level analysis of chlorophyll fluorescence and photosynthetic parameters also agreed with the expression of AtpA regulated by HRW and SNP. Altogether, our results suggested that NO might be involved in H2-improved freshness of cut lilies, and AtpA protein may play important roles during that process.

Decolorization of dyes by a novel sodium azide-resistant spore laccase from a halotolerant bacterium, Bacillus safensis sp. strain S31.

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v-1) methanol, ethanol, and acetone. In contrast to many other laccases, NaN3, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l-1) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.

Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 µM)], inhibit mitochondrial function [sodium azide (75 µM), rotenone (1 µM), berberine (100 µM), DNP (500 µM)], or directly activate AMPK [AICAR (250 µM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 µM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 µM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.