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Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.

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Raw glycerol is a byproduct of biodiesel production that currently has low to negative value for biodiesel producers. One option for increasing the value of raw glycerol is to use it as a feedstock for microbial production. Bacillus subtilis LSFM 05 was used for the production of fengycin in a mineral medium containing raw glycerol as the sole carbon source. Fengycin was isolated by acid precipitation at pH 2 and purified by silica gel column chromatography and characterized using electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) with collision-induced dissociation (CID). The mass spectrum revealed the presence of the ions of m/z 1,435.7, 1,449.9, 1,463.8, 1,477.8, 1,491.8 and 1,505.8, which were further fragmented by ESI-MS/MS. The CID profile showed the presence of a series of ions (m/z 1,080 and 966) and (m/z 1,108 and 994) that represented the different fengycin homologues A and B, respectively. Fengycin homologues A and B are variants that differ at position 6 of the peptide moiety, having either Ala or Val residues, respectively. Mass spectrometry analyses identified four fengycin A and three fengycin B variants with fatty acid components containing 14-17 carbons. These results demonstrate that raw glycerol can be used as feedstock to produce fengycin, and additional work should focus on the optimization of process conditions to increase productivity.

Assuntos

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The removal of oxidation debris from the oxidized carbon nanotube surface with a NaOH treatment is a key step for an effective functionalization and quality improvement of the carbon nanotube samples. In this work, we show via infrared spectroscopy and ultrahigh resolution and accuracy mass spectrometry that oxidation debris obtained from HNO(3)-treated multiwalled carbon nanotubes is a complex mixture of highly condensed aromatic oxygenated carbonaceous fragments. We have also evaluated their cytotoxicity by using BALB/c 3T3 mouse fibroblasts and HaCaT human keratinocytes as models. By knowing the negative aspects of dissolved organic carbon (DOC) to the water quality, we have demonstrated the removal of these carbon nanotube residues from the NaOH solution (wastewater) by using aluminium sulphate, which is a standard coagulant agent used in conventional drinking water purification and wastewater treatment plants. Our results contribute to elucidate the structural and proactive safety aspects of oxidation debris from oxidized carbon nanotubes towards a greener nanotechnology.

Assuntos

RESUMO

The deposition of mesoporous silica (SiO(2)) on carbon nanotubes (CNTs) has opened up a wide range of assembling possibilities by exploiting the sidewall of CNTs and organosilane chemistry. The resulting systems may be suitable for applications in catalysis, energy conversion, environmental chemistry, and nanomedicine. However, to promote the condensation of silicon monomers on the nanotube without producing segregated particles, (OR)(4-x)SiO(x)(x-) units must undergo nucleophilic substitution by groups localized on the CNT sidewall during the transesterification reaction. In order to achieve this preferential attachment, we have deposited silica on oxidized carbon nanotubes (single-walled and multiwalled) in a sol-gel process that also involved the use of a soft template (cetyltrimethylammonium bromide, CTAB). In contrast to the simple approach normally used to describe the attachment of inorganic compounds on CNTs, SiO(2) nucleation on the tube is a result of nucleophilic attack mainly by hydroxyl radicals, localized in a very complex surface chemical environment, where various oxygenated groups are covalently bonded to the sidewall and carboxylated carbonaceous fragments (CCFs) are adsorbed on the tubes. Si-O-C covalent bond formation in the SiO(2)-CNT hybrids was observed even after removal of the CCFs with sodium hydroxide. By adding CTAB, and increasing the temperature, time, and initial amount of the catalyst (NH(4)OH) in the synthesis, the SiO(2) coating morphology could be changed from one of nanoparticles to mesoporous shells. Concomitantly, pore ordering was achieved by increasing the amount of CTAB. Furthermore, preferential attachment on the sidewall results mostly in CNTs with uncapped ends, having sites (carboxylic acids) that can be used for further localized reactions.