Transmembrane Receptors, Cytoskeleton and Cell Cycle Genes Were Progressively Deregulated in the Bone Marrow CD19+ and CD138+ Cells of Patientswwith Waldenstrom's Macroglolubinemia (WM) Vs. Subjects with IgM Monoclonal Gammopathy of Undetermined Significance Igmmgus Vs. Healthy Subjects

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

Objective To analyze the clinical and imaging features of 2 siblings with leukoencephalopathy due to NADH dehydrogenase (ubiquinone)flavoprotein 2 (NDUFV2) gene mutation, in order to better understand and diagnose it earlier. Methods Clinical and follow-up data of the proband and his brother were collected.Clinical features including symptoms, signs and cranial magnetic resonance imaging (MRI) were analyzed, and 2 patients were followed up for a long time.Sanger sequencing, targeted next generation sequencing, and whole exome sequencing were performed to identify potential genetic variations in the 2 patients and their parents. Results (1) Clinical characteristics and follow-up: ages of onset were 4 months and 1 year respectively.Both of the patients presented rapid motor regression hyperinyotonia, positive pathological character.During the follow-up the condition became stable, motor function and cognition improved gradually after cocktail therapy.(2) Brain MRI of the 2 patients showed prominent abnormalities in deep cerebral white matter, presenting T1 hypointense, T2 and fluid attenuated inversion recovery (FLAIR) hyperintense in the periventricular area.FLAIR images revealed that the abnormal white matter was partially rarefied and cavitated.Diffusion weighted images (DWI) showed high signals along the periphery of the involved areas.The follow-up MRI showed the cavitation still existed and even expanded, and DWI showed regional linear or spotty high signals around the original lesions.(3) Novel mutations in NDUFV2 gene, c.467T>A and c. 404G>C, were identified in proband and his brother.The former inherited from his father, while the latter inherited from his mother, which was the new mutation not reported in the international. Conclusions The clinical features of the brothers presented subacute leukoencephalopathy with relatively stable or improved outcome.This was distinctive from the phenotypic features reported in 12 cases with hypertrophic cardiomyopathy or Leigh syndrome.The finding expanded the phenotypic spectrum of NDUFV2 mutations.Pathogenic gene of these patients which is the basis of genetic counseling for this family was determined. Key words: Leukoencephalopathy; Mitochondrial complex Ⅰ; NADH dehydrogenase (ubiquinone)flavoprotein 2 gene

Microbial electrosynthesis of carboxylic acids through CO2 reduction with selectively enriched biocatalyst: Microbial dynamics

Majority of animals form symbiotic relationships with bacteria. Based on the number of bacterial species associating with an animal, these symbiotic associations can be mono-specific, relatively simple (2-25 bacterial species/animal) or highly complex (>10(2)-10(3) bacterial species/animal). Photorhabdus (family-Enterobacteriaceae) forms a mono-specific symbiotic relationship with the entomopathogenic nematode Heterorhabditis. This system provides a tractable genetic model for animal-microbe symbiosis studies. Here, we investigated the bacterial factors that may be responsible for governing host specificity between nematode and their symbiont bacteria using proteomics approach. Total protein profiles of P. luminescens ssp. laumondii (host nematode- H. bacteriophora) and P. luminescens ssp. akhurstii (host nematode- H. indica) were compared using 2-D gel electrophoresis, followed by identification of differentially expressed proteins by MALDI-TOF MS. Thirty-nine unique protein spots were identified - 24 from P. luminescens ssp. laumondii and 15 from P. luminescens ssp. akhurstii. These included proteins that might be involved in determining host specificity directly (for e.g. pilin FimA, outer membrane protein A), indirectly through effect on bacterial secondary metabolism (for e.g. malate dehydrogenase Mdh, Pyruvate formate-lyase PflA, flavo protein WrbA), or in a yet unknown manner (for e.g. hypothetical proteins, transcription regulators). Further functional validation is needed to establish the role of these bacterial proteins in nematode-host specificity.

Directed Manipulation of a Flavoprotein Photocycle

To investigate the changes in protein of myocardium after hydrogen sulfide delayed preconditioning by using proteomics technology. Sixteen Sprague-Dawley rats were randomly assigned to control (group S) or hydrogen sulfide group (group H), n = 8 for each group. Myocardial ischemia/reperfusion injury model (ischemia 30 minutes followed by reperfusion 120 minutes) was reproduced at 24 hours after preconditioning either with normal saline or hydrogen sulfide for proteomics analysis in group S or group H, and the myocardial tissue was harvested. The total proteins were extracted and separated by two dimensional gel electrophoresis (2-DE), and the differential protein expression spots were analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Analysis of 2-DE showed that 929 ± 14 protein spots were found in group S and 906 ± 10 protein spots in group H, and the expression of 15 protein spots was different between two groups. These protein spots were chosen to undergo MALDI-TOF-MS analysis, and 11 proteins were preliminarily identified, including DNA ligase, cystathionine gamma-lyase, transcription initiation factor, NADH dehydrogenase, guanine nucleotide-releasing factor, fructose-bisphosphate aldolase A, glycogen synthase kinase-3, electron transfer flavoprotein subunit beta, glutathione S-transferase, soluble calcium-activated nucleotidase and S-adenosylmethionine synthetase. Hydrogen sulfide delayed preconditioning of myocardium resulted in the changes in protein expression profiles in the myocardium. The differential proteins might function as anti-oxidants, to improve the energy metabolism of myocardium, confer cytoprotection and protection of respiratory chain, thus conferring cardioprotection.

Anionπ Interactions in Flavoproteins

We investigated the role of sarcKATP channels and mitochondria in anesthetic-induced preconditioning (APC) using a knockout mouse model with deleted Kir6.2 subunit, the pore-forming part of sarcKATP channel (Kir6.2 C57BL/6 KO). Flavoprotein (FP) fluorescence, reactive oxygen species (ROS) production, and mitochondrial membrane potential, were monitored in isolated myocytes by laser scanning confocal microscopy during exposure to 1 MAC isoflurane. In the presence of isoflurane FP fluorescence increased to 180% and 190% of baseline in cardiomyocytes of WT and Kir6.2 KO mice, respectively. ROS increased to 118% and 124% of baseline in WT and Kir6.2 KO myocytes. Mitochondrial membrane potential decreased to 87% and 86% of baseline in WT and Kir6.2 KO. Oxidative stress induced cell death in 31% and 44% of WT and Kir6.2 KO. APC attenuated cell death to 21% and 35% in WT and Kir6.2 KO myocytes. Deletion of pore-forming subunit of sarcKATP channel did not alter characteristic mitochondrial responses to isoflurane in Kir6.2 KO cardiomyocytes. APC is preserved in the Kir6.2 KO cardiomyocytes, but their susceptibility to oxidative stress appears greater under basal conditions. Thus, sarcKATP channels are an important component of a cardioprotective pathway independent of mitochondria-mediated protection. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

We investigated whether differences in mitochondrial genome play a role in anesthetic-induced preconditioning (APC) in the diabetic heart model with “swapped” mitochondria. These rats have identical nuclear genome, but some bear Wistar (T2DNmtWistar) while others bear Fawn Hooded Hypertensive (T2DNmtFHH) mitochondrial genome. The production of reactive oxygen species (ROS) and changes in flavoproteins (FPs) fluorescence were monitored from ventricular myocytes in the presence of 1 MAC isoflurane by laser scanning confocal microscopy. Opening of mitochondrial permeability transition pore (mPTP) was detected by tetramethylrhodamine dye. During isoflurane, ROS were increased by 165% and 107% in T2DNmtFHH and T2DNmtWistar cardiomyocytes, respectively. In addition, isoflurane induced oxidation of mitochondrial FPs in T2DNmtFHH by 94% and by 35% in T2DNmtWistar myocytes. Compared to respective controls, APC increased the mPTP opening time in T2DNmtWistar but not in T2DNmtFHH myocytes. Our findings suggest that mitochondrial genome-related differences in the function of respiratory chain complexes have an impact on anesthetic sensitivity of the mitochondria. This in part could explain that APC protection against mPTP opening is compromised in T2DNmtFHH but not in T2DNmtWistar cardiomyocytes. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes

Early reperfusion is the best therapy for myocardial infarction (MI). Effectiveness, however, varies significantly between patients and has implications for long-term prognosis and treatment. A technique to assess the extent of myocardial salvage after reperfusion therapy would allow for high-risk patients to be identified in the early post-MI period. Mitochondrial dysfunction is associated with cell death following myocardial reperfusion and can be quantified by fluorometry. Therefore, we hypothesized that variations in the fluorescence of mitochondrial nicotinamide adenine dinucleotide (NADH) and flavoprotein (FP) can be used acutely to predict the degree of myocardial injury. Thirteen rabbits had coronary occlusion for 30 min followed by 3 h of reperfusion. To produce a spectrum of infarct sizes, six animals were infused cyclosporine A prior to ischemia. Using a specially designed fluorometric probe, NADH and FP fluorescence were measured in the ischemic area. Changes in NADH and FP fluorescence, as early as 15 min after reperfusion, correlated with postmortem assessment infarct size ( r = 0.695, p < 0.01). This correlation strengthened with time ( r = 0.827, p < 0.001 after 180 min). Clinical application of catheter-based myocardial fluorometry may provide a minimally invasive technique for assessing the early response to reperfusion therapy.

A107. Intrinsic signals of myocardial apoptosis

Fluorometry is used to detect intrinsic flavoprotein (FP) and nicotinamide adenine dinucleotide (NADH) signals in an open-chest rabbit model of myocardial ischemia-reperfusion injury. Myocyte apoptosis has been shown clinically to contribute to infarct size following reperfusion of ischemic myocardium. A noninvasive means of assessing apoptosis in this setting would aid in the treatment of subsequent ventricular remodeling. We show that in vivo fluorometry can be useful in apoptosis detection in open-chest surgeries. Specific changes in myocardial redox states have been shown to indicate the presence of apoptosis. Two main mitochondrial intrinsic fluorophores, NADH and FP signals, were measured during normoxia, ischemia, and reperfusion experimental protocol. Ischemia was induced by occlusion of the largest branch of the circumflex coronary artery and fluorescence signals are collected by applying two different fluorescence techniques: in vivo fluorometry and postmortem cryoimaging. The first technique was employed to detect FP and NADH signals in vivo and the latter technique uses freeze trapping and low-temperature fluorescence imaging. The heart is snap frozen while still in the chest cavity to make a "snapshot" of the metabolic state of the tissue. After freezing, the ischemic area and its surrounding border zone were excised and the sample was embedded in a frozen buffer for cryoscanning. These two data sets, in vivo fluorometry and low-temperature redox scanning, show consistent extreme oxidation of the mitochondrial redox states (higher redox ratio) suggesting the initiation of apoptosis following reperfusion. This represents the first attempt to assess myocyte apoptosis in the beating heart.

Optimization of crystallization of the flavoprotein WrbA by using additives

We have studied primary processes of ultrafast photoisomerization reactions of photoactive yellow protein (PYP) and visual rhodopsin (Rh) as well as ultrafast photoinduced electron transfer reactions of flavoproteins (FP) by means of the fs fluorescence up-conversion method. On the basis of these studies, we have examined the effects of the protein nanospace (PNS), where the chromophore is held, on the dynamics and mechanisms of the ultrafast and highly efficient photoinduced reactions of these proteins. In this article, we discuss mainly results of our comparative fluorescence dynamics studies on wild type (w. -t.) PYP, it’s site-directed mutants, analogues with replaced chromophore and FP, including the coherent vibrations in the ultrafast fluorescence decays of w. -t. PYP and mutants in the course of the photoisomerization, ultrafast dynamic Stokes shift of fluorescence of PYP analogues, and ultrafast electron transfer of FP.

Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein

Introduction. Mutations in the SDHD gene, a nuclear gene encoding for a mitochondrial protein involved in oxidative phosphorylation, are responsible for the majority of hereditary head and neck paragangliomas. The aim was characterization of cellular manifestations of SDHD mutations in paragangliomas. Methods. Tumours of patients with and without inherited mutations were examined using electron microscopy, enzyme histochemistry and immunohistochemistry. Results. Ultrastructurally, large numbers of mitochondria with disturbed morphology were detected. In almost all cases, the SDH enzyme activity was virtually absent. The expression of two catalytic proteins in the SDH complex displayed two general patterns: the flavo protein was strongly upregulated, whereas the iron protein was reduced in the tumour cells. However, in some cases deviations from these patterns in enzyme activity and protein expression were observed. Conclusion. Cellular aspects of SDHD mutations are composed of increased numbers of mitochondria with disturbed morphology, reduced SDH enzyme activity and aberrant expression of catalytic components of the SDH complex. In some cases, deviations from these patterns are observed. Analysis of histopathological changes will lead to further understanding of the biology of paragangliomas and contribute to clinical treatment strategies in the future.

Active oxygens are inevitably produced in chloroplats with photosynthesis. In the water-water cycle, ascorbate (AsA) is univalently oxidized to monodehydroascorbate (MDA) radical with scavenging hydrogen peroxide by AsA peroxidase and regenerated via three pathways. MDA radical reductase (MDAR), which contributes to one of these pathways, catalyzes the reduction of MDA radical to AsA using NAD(P)H as the electron donor. Cytosolic MDARs have been purified and characterized, though chloroplastic isoform has never been. We purified MDAR from stromal fraction of intact chloroplasts isolated from spinach. And then we also succeeded to purify chloroplastic isoform from crude extract of spinach leaves, which contained two major MDARs. Its molecular mass was 51 kDa as determined by SDS-PAGE, being 4 kDa larger than that of cytosolic MDAR. Its Km values for NADH and NADPH were 5 µM and 12 µM, respectively. The chloroplastic isoform showed the similar inhibition by SH-modifier and dicumarol to those of cytosolic isoform. The amino acid sequence of the chloroplastic MDAR was determined from the amino termini to 25th residues. The cDNA cloned with oligonucleotide corresponded to the amino-terminal region contained an open reading frame of 1,494 bp length. The open reading frame encoded the region expected to transit peptide consisting of 56 residues. The predicted amino acid sequence of the region expected to mature chloroplastic MDAR contains the FAD- and NAD(P)H-binding domains of flavoproteins and shows partial homology with cytosolic isozymes.

Human Mitochondrial Complex I in Health and Disease