A possible protective role of betain and omega-3 supplementation in traumatic brain injury.

INTRODUCTION: Due to irreversible damage following head trauma, many overlapping pathophysiological events occur including excitotoxicity, acidotoxicity, ionic imbalance, edema, oxidative stress inflammation and apoptosis. MATERIAL AND METHODS: In this this study, after the rats were separated in to groups theserats were fed throughout fourteen days with betaine, omega-3 or betaine+omega-3 combination in physiological limits prior to the trauma. After a closed head trauma, the damaged brain tissues were collected for biochemically and histologically analyses. This examination involved analyses of levels of caspase-3 and cytochrome C and neuron-specific enolase (NSE) levels in brain tissue. RESULTS: These analyses showed that traumatic brain injury (TBI) caused an increase in the levels of caspase-3, cytochrome C and neuron-specific enolase (NED) in the brain tissues examined. DISCUSSION: In this study, apoptotic and/or necrotic cell death via mitochondrial cytochrome C caspase pathway in traumatized cells and neuron-specific enolase (NED) increase indicative of neuronal damage confirmed the research hypothesis. CONCLUSION: Level of the biomarkers induced by brain injury in the groups fed with betaine, omega-3 and betaine+omega-3 combination before the traumatic damage approximated to that of control group values, suggesting that these products may have a neuroprotective role. KEY WORDS: Betain, Caspase-3, Cytochrome C and Neuron-specific enolase, Omega-3, Traumatic brain injury.

Microbes, cables, and an electrical touch

In nature, highly efficient and diverse consortia of microbes cycle carbon and other elements while generating energy for growth. Driving these reactions are organisms with the ability to extract electrons from the chemical substrates and transfer them to insoluble and soluble electron acceptors. One bacterial group in particular, Geobacter spp., can couple their respiratory metabolism to the reduction of insoluble minerals, such as iron and manganese oxides, and soluble toxic metals such as uranium. Key to these activities is the ability of the cells to transfer respiratory electrons extracellularly using an electroactive cell envelope containing abundant metalloproteins, including c-cytochromes, and conductive protein appendages or pili (known as nanowires). Thus, in addition to been ecological drivers of the cycling of carbon and metals in nature, these organisms show promise for the bioremediation of environments impacted with toxic metals. The electrical activity of Geobacter can also be mimicked in electrochemical reactors equipped with an electrode poised at a metabolically oxidizing potential, so that the electrode functions as an unlimited sink of electrons to drive the oxidation of electron donors and support cell growth. Electrochemical reactors are promising for the treatments of agricultural, industrial, and human wastes, and the electroactivity of these microbes can be used to develop materials and devices for bioenergy and bioremediation applications (AU)

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Complementary surface-enhanced resonance Raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles.

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO(2) amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Identification of c-type heme-containing peptides using nonactivated immobilized metal affinity chromatography resin enrichment and higher-energy collisional dissociation.

The c-type cytochromes play essential roles in many biological activities of both prokaryotic and eukaryotic cells, including electron transfer, enzyme catalysis, and induction of apoptosis. We report a novel enrichment strategy for identifying c-type heme-containing peptides that uses nonactivated IMAC resin. The strategy demonstrated at least 7-fold enrichment for heme-containing peptides digested from a cytochrome c protein standard, and quantitative linear performance was also assessed for heme-containing peptide enrichment. Heme-containing peptides extracted from the periplasmic fraction of Shewanella oneidensis MR-1 were further identified using higher-energy collisional dissociation tandem mass spectrometry. The results demonstrated the applicability of this enrichment strategy to identify c-type heme-containing peptides from a highly complex biological sample and, at the same time, confirmed the periplasmic localization of heme-containing proteins during suboxic respiration activities of S. oneidensis MR-1.

Identification and characterization of UndAHRCR-6, an outer membrane endecaheme c-type cytochrome of Shewanella sp. strain HRCR-6.

UndA(HRCR-6) was identified from the metal-reducing bacterium Shewanella sp. strain HRCR-6. Both in vivo and in vitro characterization results indicate that UndA(HRCR-6) is an outer membrane endecaheme c-type cytochrome and probably has a key functional role in the extracellular reduction of iron [Fe(III)] oxides and uranium [U(VI)] by Shewanella sp. HRCR-6.

Opportunities for mesoporous nanocrystalline SnO2 electrodes in kinetic and catalytic analyses of redox proteins.

PFV (protein film voltammetry) allows kinetic analysis of redox and coupled-chemical events. However, the voltammograms report on the electron transfer through a flow of electrical current such that simultaneous spectroscopy is required for chemical insights into the species involved. Mesoporous nanocrystalline SnO(2) electrodes provide opportunities for such 'spectroelectrochemical' analyses through their high surface area and optical transparency at visible wavelengths. Here, we illustrate kinetic and mechanistic insights that may be afforded by working with such electrodes through studies of Escherichia coli NrfA, a pentahaem cytochrome with nitrite and nitric oxide reductase activities. In addition, we demonstrate that the ability to characterize electrocatalytically active protein films by MCD (magnetic circular dichroism) spectroscopy is an advance that should ultimately assist our efforts to resolve catalytic intermediates in many redox enzymes.

Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1.

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe(2)O(3)), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Trace analysis of proteins using postseparation solution-phase digestion and electrospray mass spectrometric detection of marker peptides.

Analytical methodologies for the absolute quantitation of proteins typically include a digest step often using trypsin as the proteolytic enzyme. In the majority of cases, off-line and on-line digestion methods are implemented prior to an LC-MS analysis system, requiring a high sequence coverage for unambiguous protein identification. For proteins with a strong overlap in amino acid sequence, e.g., therapeutic proteins and their metabolites, it is essential to separate proteins prior to digestion and the subsequent electrospray mass spectrometry analysis of marker peptides. Here, we present an on-line postcolumn solution-phase digestion methodology that is based on the continuous infusion of the proteolytic enzyme pepsin downstream to the nano C18 reversed-phase column. Proteins are identified based on their retention time in combination with the detection of specific marker peptides formed in the postcolumn digest. The optimization of important parameters such as enzyme concentration, reaction time, and organic modifier concentration is described. We demonstrated that the continuous-flow solution-phase digest method can be coupled on-line to the reversed-phase gradient liquid chromatography separation of proteins. Detection limits obtained for five model proteins, detected as specific marker peptides with m/z values of 300-1000, range from 30 to 90 fmol, with a linear response up to 3 pmol.

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions.

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.

Redox infrared markers of the heme and axial ligands in microperoxidase: Bases for the analysis of c-type cytochromes.

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.

Relationship between immune status and activity of the lymphocyte energy supply system in adolescents suffering from frequent diseases.

Children and adolescents aged 4-16 years with the diagnosis of acute respiratory viral infection with long-lasting fever, manifestations of intoxication syndrome, and catarrhal symptoms were examined. In children and adolescents suffering from frequent diseases and presented with acute respiratory viral infection we found disorders in the immune status (depression of the cellular component, helper/suppressor imbalance, suppressed production of IgA and hyperproduction of IgM, decreased concentration of secretory IgA in the saliva) in comparison with children rarely falling ill. The redox potential and lymphocyte cytochrome C content were decreased in adolescents often falling ill, while the content of cytochrome oxidase did not change. A negative multiple correlation (R=6.8, p<0.005) was detected between the decrease in cytochrome C content and NADP/NADPH redox potential and increase in the immunoregulatory index. ATP content in lymphocyte from adolescents frequently falling ill remained 21% decreased during the first 2 weeks after acute respiratory viral infection, while the ATP/ADP ratio was shifted towards dinucleotide, which also indicated disorders in ATP synthesis in lymphocytes.

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry.

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.

Indirect effects of Bax and Bak initiate the mitochondrial alterations that lead to cytochrome c release during arsenic trioxide-induced apoptosis.

Arsenic trioxide is a potent chemotherapeutic agent by virtue of its ability to selectively trigger apoptosis in tumor cells. Previous studies have demonstrated that arsenicals cause direct damage to mitochondria, but it is not clear that these effects initiate apoptosis. Here we used Bak-/- mouse liver mitochondria and virally immortalized Bax-/- Bak-/- mouse embryonic fibroblasts (MEFs) to investigate whether or not multidomain proapoptotic BCL-2 family proteins were required for arsenic-induced mitochondrial damage and cell death. At clinically achievable concentrations, arsenic stimulated cytochrome c release and apoptosis via a Bax/Bak-dependent mechanism. At higher concentrations (125 microM-1 mM), cells died via a Bax/Bak-independent mechanism mediated by oxidative stress that resulted in necrosis. Consistent with previous reports, arsenic directly inhibited complex I of the mitochondrial electron transport chain, which resulted in mitochondrial permeability transition (MPT), accompanying generation of reactive oxygen species (ROS), and thiol oxidation. However, these effects only occurred at concentrations of arsenic trioxide of 50 microM and higher, and the oxidative stress associated with these effects blocked caspase activation. Our data demonstrate for the first time that the cytochrome c release which initiates apoptosis in cells exposed to this classic mitochondrial poison occurs indirectly via the activation of Bax/Bak rather than via direct mitochondrial damage. Furthermore, the results implicate reactive oxygen species in a concentration-dependent mechanistic switch between apoptosis and necrosis.

Progression of hair cell ejection and molecular markers of apoptosis in the avian cochlea following gentamicin treatment.

Aminoglycoside treatment induces caspase-dependent apoptotic death in inner ear sensory hair cells. The timing of apoptotic signaling in sensory hair cells following systemic aminoglycoside treatment has not been characterized in vivo. We administered a single subcutaneous injection of the aminoglycoside gentamicin (300 mg/kg) to 12-16-day-old chicks and used immunocytochemical techniques to document the following responses in affected hair cells: T-cell restricted intracellular antigen-related protein (TIAR) translocation from the nucleus to the cytoplasm, cytochrome c release from the mitochondria, caspase-3 activation, nuclear condensation, and an orderly progression of hair cell ejection from the proximal end of the basilar papilla. Hair cells in the proximal tip exhibited TIAR translocation from the nucleus and aggregation into punctate granules in the cytoplasm 12 hours after injection and the response progressed distally. Cytochrome c release from the mitochondria into the cytoplasm and caspase-3 activation were observed in affected hair cells immediately prior to and during ejection. Hair cell ejection occurred between 30 and 54 hours after injection, beginning in the proximal tip and progressing distally. Nuclear condensation accompanied ejection while the loss of: 1) membrane integrity; 2) phalloidin labeling of F-actin; and 3) TO-PRO-1 labeling of nuclear contents occurred within 48 hours following ejection. Our results present a timeline of aminoglycoside-induced inner ear sensory hair cell apoptotic death that includes an 18-hour window between the initial apoptotic response and the later stages of programmed death signaling that accompany ejection and a gradual breakdown of hair cells following ejection.

A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase.

In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550). The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins. We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion.

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.

The proteasome inhibitor bortezomib promotes mitochondrial injury and apoptosis induced by the small molecule Bcl-2 inhibitor HA14-1 in multiple myeloma cells.

Interactions between the small molecule Bcl-2 inhibitor HA14-1 and proteasome inhibitors, including bortezomib (Velcade; formerly known as PS-341) and MG-132, have been examined in human multiple myeloma cells. Sequential (but not simultaneous) exposure of MM.1S cells to bortezomib or MG-132 (10 h) followed by HA14-1 (8 h) resulted in a marked increase in mitochondrial injury (loss of DeltaPsim, cytochrome c, Smac/DIABLO, and apoptosis-inducing factor release), activation of procaspases-3, -8, and -9, and Bid, induction of apoptosis, and loss of clonogenicity. Similar interactions were observed in U266 and MM.1R dexamethasone-resistant myeloma cells. These events were associated with Bcl-2 cleavage, Bax, Bak, and Bad accumulation, mitochondrial translocation of Bax, abrogation of Mcl-1, Bcl-xL, and XIAP upregulation, and a marked induction of JNK and p53. Bortezomib/HA14-1 treatment triggered an increase in reactive oxygen species (ROS), which, along with apoptosis, was blocked by the free radical scavenger N-acetyl-L-cysteine (L-NAC). L-NAC also opposed bortezomib/HA14-1-mediated JNK activation, upregulation of p53 and Bax, and release of cytochrome c and Smac/DIABLO. Finally, bortezomib/HA14-1-mediated apoptosis was unaffected by exogenous IL-6. Together, these findings indicate that sequential exposure of myeloma cells to proteasome and small molecule Bcl-2 inhibitors such as HA14-1 may represent a novel therapeutic strategy in myeloma.

Sensory neurons with activated caspase-3 survive long-term experimental diabetes.

Long-term experimental diabetes may best model the prominent and irreversible sensory deficits of chronic human diabetic polyneuropathy. Whereas irretrievable loss of sensory neurons, if present, would be an unfortunate feature of the disease, systematic unbiased counting has indicated that sensory neurons survive long-term experimental diabetes. In this study, we examined whether incipient cell loss from apoptosis in chronic experimental diabetes might nonetheless be in process, or whether neurons somehow adapt to their chronic insults. We examined sensory neurons in L4 and L5 dorsal root ganglia of long-term experimental streptozotocin-induced diabetic rats using transferase-mediated dUTP nick-end labeling (TUNEL), 4',6-diamidino-2-phenylindole (DAPI) staining of nuclear morphology, and electron microscopic appraisal of cell morphology. None provided any evidence for ongoing apoptosis. Despite this confirmation that sensory neurons survive, neurons had elevated expression of activated caspase-3 in unique patterns that included their nuclei, cytoplasm, and proximal axonal segments. Bcl-2 expression, a marker of antiapoptosis signaling, was observed in similar numbers of diabetic and nondiabetic neurons. In contrast, diabetic sensory neurons had elevated expression of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) in their nuclei, cytoplasm, and proximal axonal segments not overlapping with caspase-3 localization. Diabetic sensory neurons also had an apparent rise in cytoplasmic labeling of nitrotyrosine, a marker of peroxynitrite toxicity reported to activate PARP.