Protein Cytochrome Research Articles

MicroRNA-8126-Mediated Antioxidant Stress Attenuates Isoflurane-Induced Hippocampal Neurotoxicity in Developing Rats.

Objective To investigate the effect of microRNA-8126 (miR-8126) on isoflurane-induced hippocampal neurotoxicity in rats. Methods A rat isoflurane nerve injury model was constructed. The expression of miR-8126 in the hippocampal region of normal and injured rats was measured by qRT-PCR; synaptic density protein-95, PAK-3 (p21-activated kinase-3) and apoptosis-related proteins cytochrome C, cleaved caspase-3, and cleaved PARP were detected by Western blot. The Cytochrome C, cleaved-caspase-3, and cleaved PARP expression was detected by WB, as well as GSH-Px, CAT, SOD, and ROS. Results miR-8126 was lowly expressed in the isoflurane-treated rat hippocampal region and in rat hippocampal neuronal cells, and the expression of apoptosis-related proteins and apoptosis levels were significantly increased, and neural activity, cell activity, and proliferation capacity were significantly decreased. Oxidative stress levels and ROS content were significantly increased; overexpression of miR-8126 in the rat hippocampal region significantly inhibited oxidative stress and apoptosis. Overexpression of miR-8126 in rat hippocampal neural progenitor cells significantly increased cell activity, proliferative capacity, and significantly smaller mitochondrial size and it decreased ROS content and oxidative stress levels and apoptosis-related protein expression compared to isoflurane-treated cells; while inhibition of miR-8126 expression in rat hippocampal neuronal cells significantly decreased cell activity, proliferative capacity, and mitochondrial size compared to the control group. In contrast, inhibition of miR-8126 expression in rat hippocampal neuronal cells resulted in a further decrease in cell activity, proliferation capacity, and significantly larger mitochondrial size and increased expression of apoptosis-related proteins compared with the control group. miR-8126 regulates the activity of rat hippocampal neuronal cells by targeting ATF4. Conclusions miR-8126 attenuates isoflurane-induced hippocampal neurotoxicity in rats by mediating antioxidative stress.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

Combined use of phosphonium-erythrosin B-based nanoGUMBOS, UV–Vis spectroscopy, and chemometrics for discrimination and quantification of proteins

Nanoparticles derived from a group of uniform materials based on organic salts (nanoGUMBOS) are considered promising candidates for protein analysis due to their facile synthesis in aqueous media and high tunability. In this study, a phosphonium-erythrosin B-based nanoGUMBOS (i.e., [P4444]2[EB]) was prepared using an ultrasound-assisted reprecipitation method, and its ability to discriminate and quantify proteins was evaluated. Sonication time (30 s, 5 min, and 15 min) and cyclodextrin templating (α-, 2-HP-β-, and γ-CD) were investigated for their effects on discrimination performance of synthesized nanomaterial. Six proteins (albumin, hemoglobin, trypsin, catalase, lysozyme, and cytochrome c) with different abundance levels and physicochemical properties were selected as target analytes. Absorbance response patterns generated from interactions between [P4444]2[EB] nanoGUMBOS and proteins were analyzed using partial least squares discriminant analysis. Percentages of correct protein discrimination ranged from 94.6 to 99.6%, with the latter being the best result obtained using non-templated nanoGUMBOS formed after 5 min sonication. Under optimized conditions, it was possible to discriminate all protein samples with percentages of correct assignments greater than 90% for concentrations as low as 2.0 μg mL−1. The discrimination capability of synthesized nanoGUMBOS was further evaluated using mixtures of different ratios of lysozyme, cytochrome c, and hemoglobin. Finally, partial least squares models were developed for protein quantification and the best performance was observed for albumin. Results support potential use of [P4444]2[EB] nanoGUMBOS in combination with ultraviolet–visible spectroscopy and chemometrics for qualitative and quantitative analyses of individual proteins and mixtures of proteins.

Protective effect and mechanism of SeMet-Pro-Ser on K2Cr2O7-induced toxicity in L-02 hepatocytes

SeMet-Pro-Ser (Se-MPS), identified from selenium-enriched brown rice protein hydrolysates, shows strong free radical scavenging capacities and hexavalent chromium [Cr(VI)] reduction activity. This study explored the protective effect and mechanism of Se-MPS on potassium dichromate (K2Cr2O7)-induced toxicity in L-02 hepatocytes. Exposure of L-02 hepatocytes to K2Cr2O7 drastically decreased the cell viability and caused cell apoptosis, which was effectively suppressed by Se-MPS treatment in a dose-dependent manner. Se-MPS significantly prevented the increase of reactive oxygen species (ROS) and malondialdehyde (MDA) generation, the decrease of antioxidant enzyme activities; and the depletion of reduced glutathione (GSH) induced by K2Cr2O7. In addition, Se-MPS could alleviate K2Cr2O7- induced apoptosis as assessed by mitochondrial membrane potential and caspase family. The expression levels of apoptosis-related proteins Bax and cytochrome C were significantly downregulated, and anti-apoptotic protein Bcl-2 was significantly upregulated in the Se-MPS plus K2Cr2O7 group. This study provided further insights into Se-MPS in resisting K2Cr2O7-induced cytotoxicity.

Next generation VAMS®–Trypsin immobilization for instant proteolysis in bottom-up protein determination

• The tedious tryptic digestion step for proteomic analyses is incorporated in VAMS® samplers. • Smart proteolysis sampling initiates preparation already at sample collection. • Trypsin is covalently bound to VAMS® which than serves as smart sampler. • Smart sampler can be applied in a proteomic workflow. • Proteins in complex biological matrices like human serum are digested. The purpose of this study was to develop a smart proteolysis sampler based on commercially available VAMS® tips employing Volumetric Absorptive Microsampling. In this proof-of-concept paper we show that covalently bound trypsin to VAMS exhibits sufficient activity for proteomic purposes without altering physical conditions. Alkaline pH was needed for the covalent binding of trypsin and at least 50 μg trypsin was required to obtain maximal activity. Prolonged digestion times (up to overnight) of a sample containing 7 model proteins on the VAMS yielded higher peak intensities for signature peptides which are beneficial for quantitative analysis. In-solution digestions of three proteins (carbonic anhydrase 2, catalase and BSA) yielded higher intensities of their signature peptides whereas the other four proteins (cytochrome C, myoglobin, β-lactoglobulin, and phosphorylase B) showed higher intensities for their signature peptides from digestions performed on trypsin modified VAMS. In comparison with conventional in-solution digestions, approximately half of the proteins were identified from diluted serum using trypsin on VAMS. For shotgun serum proteomics, diluted samples collected on trypsin modified VAMS followed by immediate drying yielded similar peptide- (942 vs 875) and protein (96 vs 99) identifications compared to those collected on trypsin modified VAMS followed by overnight digestions. This result shows the potential of trypsin on VAMS digestions.

A murine model of ischemia–reperfusion: the perfusion with leptin promotes the apoptosis-related relocation of mitochondrial proteins Bax and cytochrome c

BackgroundLeptin exerts both protective and deleterious effects on the heart; the first occurs under hypoxia- or ischemia-associated damage, the second is a pro-hypertrophic factor on cardiomyocytes. Therefore, leptin could represent a link between obesity and cardiovascular diseases. The study aimed to investigate the effect of leptin—the same concentration that is frequently measured in obesity and induces cardiac hypertrophy—on murine hearts following an episode of ischemia–reperfusion; moreover, we evaluated the heart's performance, hypertrophy, and activation of apoptosis. Rat hearts were perfused continuously with or without 3.1 nM leptin for one h before and 1 h after an episode of ischemia. Cardiac performance was evaluated, homogenates and mitochondria were prepared for western blot analysis of cardiac actin, leptin receptor, STAT3, pSTAT3, and apoptosis-related proteins Bax, Bcl-2, cytochrome c, and caspase 3.ResultsLeptin worsened heart recovery after ischemia (p < 0.05 Control vs IR + Lep of Heart Perform, Fig. 2). Although no hypertrophic response was observed, leptin induced the migration of Bax to the mitochondria and the release of cytochrome c into the cytosol (p < 0.05 Control vs IR + Lep, Fig. 5), essential events in the intrinsic/mitochondrial apoptosis.ConclusionsOur results indicate that the presence of leptin for 1 h before and after the ischemic insult reduces heart recovery and amplifies apoptotic signaling through the mitochondrial pathway.

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.

Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation.

In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.

Decreased Expression of CIRP Induced by Therapeutic Hypothermia Correlates with Reduced Early Brain Injury after Subarachnoid Hemorrhage.

Early brain injury is considered to be a primary reason for the poor prognosis of patients suffering from subarachnoid hemorrhage (SAH). Due to its pro-inflammatory activity, cold-inducible RNA-binding protein (CIRP) has been implicated in the ischemic brain insult, but its possible interplay with hypothermia in SAH treatment remains to be evaluated. One-hundred and thirty-eight Sprague-Dawley rats (300–350 g males) were randomly allocated into the following groups: sham-operated (Sham); SAH; and SAH + hypothermia (SAH + H), each comprised of 46 animals. After treatments, the brain tissues of the three groups were randomly collected after 12 h, 1 d, 3 d, and 7 d, and the expression levels of the CIRP and mitochondrial apoptosis pathway-related proteins Bax, Bcl-2, caspase-9, caspase-3, and cytochrome c measured using Western blotting and real-time PCR. Brain damage was assessed by TUNEL and Nissl staining, the electron microscopy of brain tissue slices as well as functional rotarod tests. Expression of CIRP, Bax, caspase-9, caspase-3, and cytochrome c as well as reduced motor function incidence were higher in the SAH group, particularly during the first 3 d after SAH induction. Hypothermia blunted these SAH responses and apoptosis, thereby indicating reduced inflammatory signaling and less brain cell injury in the early period after SAH. Hypothermia treatment was found to effectively protect the brain tissue from early SAH injury in a rat model and its further evaluation as a therapeutic modality in SAH patients requires further study.

Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

Preparation of NIR-Responsive Gold Nanocages as Efficient Carrier for Controlling Release of EGCG in Anticancer Application.

Hepatocellular carcinoma (HCC) is a type of cancer that has a restricted therapy option. Epigallocatechin gallate (EGCG) is one of the main biologically active ingredients in tea. A large number of studies have shown that EGCG has preventive and therapeutic effects on various tumors. In addition, the development of near-infrared (NIR)-responsive nano-platforms has been attracting cancer treatment. In this work, we designed and synthesized a strategy of gold nanocages (AuNCs) as an efficient carrier for controlling release of EGCG for anti-tumor to achieve the synergistic functions of NIR-response and inhibited tumor cell proliferation. The diameter of AuNCs is about 50 nm and has a hollow porous (8 nm) structure. Thermal imaging-graphic studies proved that the AuNCs-EGCG obtained have photothermal response to laser irradiation under near-infrared light and still maintain light stability after multiple cycles of laser irradiation. The resulted AuNCs-EGCG reduced the proliferation rate of HepG2 cells to 50% at 48 h. Western blot analysis showed that NIR-responsive AuNCs-EGCG can promote the expression of HepG2 cell apoptosis-related proteins HSP70, Cytochrome C, Caspase-9, Caspase-3, and Bax, while the expression of Bcl-2 is inhibited. Cell confocal microscopy analysis proved that AuNCs-EGCG irradiated by NIR significantly upregulates Caspase-3 by nearly 2-fold and downregulates Bcl-2 by nearly 0.33-fold, which is beneficial to promote HepG2 cell apoptosis. This study provides useful information for the NIR-responsive AuNCs-EGCG as a new type of nanomedicine for HCC.

Danggui Buxue Decoction improves heart function of chronic intermittent hypoxia mice by promoting mitochondrial autophagy and inhibiting cardiomyocyte apoptosis

The study established a chronic intermittent hypoxia(CIH) model in mice to investigate the effects of Danggui Buxue Decoction(DBD) on mitochondrial autophagy and cardiomyocyte apoptosis and explore its protective effect and mechanism on cardiac function of CIH mice. Forty C57 BL/6 N male mice were randomly divided into the control(CON) group, CIH group, CIH+DBD group, and DBD group, with 10 mice in each group. CIH was induced by filling the hypoxic chamber with N_2(90 s) to reduce the O_2 concentration to 5% and then filling the hypoxic chamber with O_2(90 s) to restore O_2 concentration to 21%, 3 min per cycle, and the CIH treatment continued for 35 d, 8 h per day. Mice in the CIH+DBD and DBD groups were treated with intragastric administration of DBD every day, while those in the CON and CIH groups with the same volume of normal saline. The cardiac function of mice was measured by echocardiography. The pathological changes in myocardium were observed after HE staining, followed by the observation of cardiomyocyte apoptosis by Tunel staining. The expression of apoptosis-related proteins pro-caspase-3, caspase-3, Bcl-2, and Bax and autophagy-related proteins LC3Ⅱ, LC3Ⅰ, P62, parkin, and cytochrome C(Cytc) was detected by Western blot. The mitochondrial membrane potential was observed using JC-1 fluorescent probe. Compared with the CON group, the CIH group exhibited remar-kably lowered left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), elevated left ventricular end-systolic volume(LVESV) and end-diastolic volume(LVEDV), disordered myocardial fiber arrangement, increased number of TUNEL-positive cells, decreased pro-caspase-3/caspase-3, Bcl-2/Bax, and LC3Ⅱ/LC3Ⅰ ratios, parkin, mitochondrial Cytc expression, and mitochondrial membrane potential, and up-regulated P62 and Cytc expression. Compared with the CIH group, DBD increased LVEF, LVFS, pro-caspase-3/caspase-3, Bcl-2/Bax, and LC3Ⅱ/LC3Ⅰ ratios, and parkin expression, as well as mitochond-rial Cytc expression, and mitochondrial membrane potential, decreased LVESV, LVEDV, and the number of Tunel-positive cells, and improved the myocardial fiber arrangement. DBD has a protective effect on the heart function of CIH mice. It improves the heart function possibly by promoting mitochondrial autophagy to ameliorate mitochondrial function and inhibiting the cardiomyocyte apoptosis.

Comparative analysis of hydration layer reorientation dynamics of antifreeze protein and protein cytochrome P450

Antifreeze proteins (AFPs) inhibit ice re-crystallization by a mechanism remaining largely elusive. Dynamics of AFPs’ hydration water and its involvement in the antifreeze activity have not been identified conclusively. We herein, by simulation and theory, examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm, Choristoneura fumiferana, compared with a protein cytochrome P450 (CYP). The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules. Therefore, the jump reorientation of water molecules around the AFP active region is accelerated. Due to the mutual coupling and excitation of hydrogen bond exchange, with the acceleration of hydrogen bond exchange, the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated. Therefore, the water reorientation dynamics of AFP is faster than that of CYP. The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.

Catalytic electrochemistry of the bacterial Molybdoenzyme YcbX

Molybdenum-dependent enzymes that can reduce N-hydroxylated substrates (e.g. N-hydroxyl-purines, amidoximes) are found in bacteria, plants and vertebrates. They are involved in the conversion of a wide range of N-hydroxylated organic compounds into their corresponding amines, and utilize various redox proteins (cytochrome b5, cyt b5 reductase, flavin reductase) to deliver reducing equivalents to the catalytic centre. Here we present catalytic electrochemistry of the bacterial enzyme YcbX from Escherichia coli utilizing the synthetic electron transfer mediator methyl viologen (MV2+). The electrochemically reduced form (MV+.) acts as an effective electron donor for YcbX. To immobilize YcbX on a glassy carbon electrode, a facile protein crosslinking approach was used with the crosslinker glutaraldehyde (GTA). The YcbX-modified electrode showed a catalytic response for the reduction of a broad range of N-hydroxylated substrates. The catalytic activity of YcbX was examined at different pH values exhibiting an optimum at pH 7.5 and a bell-shaped pH profile with deactivation through deprotonation (pKa1 9.1) or protonation (pKa2 6.1). Electrochemical simulation was employed to obtain new biochemical data for YcbX, in its reaction with methyl viologen and the organic substrates 6-N-hydroxylaminopurine (6-HAP) and benzamidoxime (BA).

Septins promote caspase activity and coordinate mitochondrial apoptosis.

Apoptosis is a form of regulated cell death essential for tissue homeostasis and embryonic development. Apoptosis also plays a key role during bacterial infection, yet some intracellular bacterial pathogens (such as Shigella flexneri, whose lipopolysaccharide can block apoptosis) can manipulate cell death programs as an important survival strategy. Septins are a component of the cytoskeleton essential for mitochondrial dynamics and host defense, however, the role of septins in regulated cell death is mostly unknown. Here, we discover that septins promote mitochondrial (i.e., intrinsic) apoptosis in response to treatment with staurosporine (a pan-kinase inhibitor) or etoposide (a DNA topoisomerase inhibitor). Consistent with a role for septins in mitochondrial dynamics, septins promote the release of mitochondrial protein cytochrome c in apoptotic cells and are required for the proteolytic activation of caspase-3, caspase-7, and caspase-9 (core components of the apoptotic machinery). Apoptosis of HeLa cells induced in response to infection by S. flexneri ΔgalU (a lipopolysaccharide mutant unable to block apoptosis) is also septin-dependent. In vivo, zebrafish larvae are significantly more susceptible to infection with S. flexneri ΔgalU (as compared to infection with wildtype S. flexneri), yet septin deficient larvae are equally susceptible to infection with S. flexneri ΔgalU and wildtype S. flexneri. These data provide a new molecular framework to understand the complexity of mitochondrial apoptosis and its ability to combat bacterial infection.

Proteomic analysis of a copper mine isolated fungus Rhizopus microspores IOC 4686 when exposed to copper sulfate

The fungus Rhizopus microsporus, known for its absorption capacity for toxic metals was used to understand the green metal recovery via analysis of its physiology under metal stress conditions through proteomic methods. To investigate the effects of copper stress on fungus, R. microsporus IOC 4686, isolated from the mine environment, was exposed to copper ions (50mgL-1) for 48 h. This study was performed only on copper exposure. Tryptic and chymotryptic extracts of proteins were analyzed by nano- liquid chromatography-tandem mass spectrometry, identification was performed by PEAKS Studio 8.5. Proteins were classified according to their molecular function and biological process. Enzymes, such as catalase, superoxide dismutase, and cytochrome c peroxidase were found in the presence and absence of copper ions. However, only in presence of copper ions, was observed the presence of heat shock proteins (HPS 20, HPS 70, and HPS 78) and metalloproteins (GrpE protein homolog and cytochrome P450). These classes of proteins have been produced by cells in response to stress conditions. The control group (absence of copper ions) also presented antioxidant enzymes suggesting that the fungus isolated from the mine environment already has adapted to the copper. The presence of these proteins suggested a physiological response of R. microsporus IOC 4686 to oxidative stress induced by copper.

Low morphological and genetic variation within the glass-perchlet Parambassis siamensis (Teleostei: Ambassidae) in Peninsular Malaysia.

We compare several populations of the glass-perchlet Parambassis siamensis (Fowler 1937) (Teleostei: Ambassidae) sampled throughout Peninsular Malaysia to determine their degree of differentiation, using both morphological and molecular characters. Our morphological analyses do not show evidence for the presence of more than one species, with the range of morphometric and meristic characters overlapping among populations. Our genetic analysis using partial sequences of the mitochondrial gene coding for the protein cytochrome c oxidase I (COI) reveals the existence of two clades that diverge from each other by a minimum uncorrected p-distance of 2.2%. The first clade comprises of specimens from south-eastern Peninsular Malaysia (Pahang and Endau-Rompin River basins) along with those from Cambodia (lower Mekong River). The second clade comprises of specimens from western Peninsular Malaysia (Selangor, Kurau, Perak, Muda and Kerian River basins) and north-eastern Peninsular Malaysia (Terengganu River basin), along with those from Chao Phraya River basin nearby Bangkok (type locality of P. siamensis). The presence of specimens with numerous melanophores on body sides in each of these two clades indicates that body marking pattern is not a valuable taxonomic character. This finding supports the conclusion that Chanda punctulata Fraser-Brunner 1955 is a junior synonym of P. siamensis. Altogether, our results support the hypothesis that all populations of P. siamensis in Peninsular Malaysia (along those from Chao Phraya and lower Mekong basins) are conspecific, comprising two genetically distinct, although close lineages. We further discuss the phenotypic plasticity within P. siamensis in relation to lotic and lentic habitats. Finally, we briefly discuss some implications for biogeography and possible causes explaining the distribution pattern.

Detergent-Free Functionalization of Hybrid Vesicles with Membrane Proteins Using SMALPs.

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo3 and can also be performed with complex membrane protein mixtures.

A Novel Imidazo[1,2-a]pyridine Compound Reduces Cell Viability and Induces Apoptosis of HeLa Cells by p53/Bax-Mediated Activation of Mitochondrial Pathway.

Despite emerging research on new treatment strategies, chemotherapy remains one of the most important therapeutic modalities for cancers. Imidazopyridines are important targets in organic chemistry and, given their numerous applications, they are worthy of attention. The objective of this study was to design and synthesize a novel series of imidazo[1,2-a]pyridine-derived compounds and investigate their antitumor effects and the underlying mechanisms. Imidazo[1,2-a]pyridine-derived compounds were synthesized with new strategies and conventional methods. The antitumor activities of the new compounds were evaluated by MTT assay. Flow cytometry and immunofluorescence were performed to examine the effects of the most effective antiproliferative compound on cell apoptosis. Western blot analysis was used to assess the expression of apoptotic proteins. Fifty-two new imidazo[1,2-a]pyridine compounds were designed and successfully synthesized. The compound, 1-(imidazo[1,2-a]pyridin-3-yl)-2-(naphthalen-2-yl)ethane-1,2-dione, named La23, showed high potential for suppressing the viability of HeLa cells (IC50 15.32 μM). La23 inhibited cell proliferation by inducing cell apoptosis, and it reduced the mitochondrial membrane potential of HeLa cells. Moreover, treatment with La23 appeared to increase the expression of apoptotic-related protein P53, Bax, cleaved caspase-3, and cytochrome c at a low concentration range. The novel imidazo[1,2-a]pyridine compound, La23, was synthesized and it suppressed cell growth by inducing cell apoptosis via the p53/Bax mitochondrial apoptotic pathway.

Proteome profiling reveals changes in energy metabolism, transport and antioxidation during drought stress in Nostoc flagelliforme

BackgroundDrought is an important abiotic stress that constrains the growth of many species. Despite extensive study in model organisms, the underlying mechanisms of drought tolerance in Nostoc flagelliforme remain elusive.ResultsWe characterized the drought adaptation of N. flagelliforme by a combination of proteomics and qRT-PCR. A total of 351 differentially expressed proteins involved in drought stress adaptation were identified. It was found that the expression of several nutrient influx transporters was increased, including molybdate ABC transporter substrate binding protein (modA), sulfate ABC transporter substrate-binding protein (sbp) and nitrate ABC transporter (ntrB), while that of efflux transporters for toxic substances was also increased, including arsenic transporting ATPase (ArsA), potassium transporter (TrkA) and iron ABC transporter substrate-binding protein (VacB). Additionally, photosynthetic components were reduced while sugars built up during drought stress. Non-enzymatic antioxidants, orange carotenoid protein (OCP) homologs, cytochrome P450 (CYP450), proline (Pro) and ascorbic acid (AsA) were all altered during drought stress and may play important roles in scavenging reactive oxygen species (ROS).ConclusionIn this study, N. flagelliforme may regulates its adaptation to drought stress through the changes of protein expression in photosynthesis, energy metabolism, transport, protein synthesis and degradation and antioxidation.Highlights• A total of 351 DEPs involved in adaptation to drought stress were identified.• Changes in the expression of six OCP homologs were found in response to drought stress.• Differential expression of transporters played an important role in drought stress adaptation.• Most PSII proteins were downregulated, while PSI proteins were unchanged in response to drought stress.• Sugar metabolism was upregulated in response to drought stress.