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The endoplasmic reticulum (ER) is responsible for processing of proteins that are destined to be secreted, enclosed in a vesicle, or incorporated in the plasma membrane. Nascent peptides that enter the ER undergo a series of highly regulated processing steps to reach maturation as they transit the ER. Alterations in the intracellular environment that induce ER stress are thought to interrupt these processing steps, and result in unfolding of proteins in the ER. Accumulation of unfolded proteins concurrently activates three transmembrane stress sensors, IRE1, ATF6 and PERK, and is referred to as the Unfolded Protein Response (UPR). Our understanding of the mechanisms of UPR induction has been assembled primarily from experiments inducing ER stress with chemical and genetic manipulations. However, physiological stress often induces activation of ER stress sensors in a distinct manner from the canonical UPR. The unique activation profiles in vivo have prompted us to examine the mechanism of UPR activation in neurons following cerebral ischemia. [ABSTRACT FROM AUTHOR]

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (- 14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma. [ABSTRACT FROM AUTHOR]

The critical event of the intrinsic pathway of apoptosis following transient global brain ischemia is the release of cytochrome c from the mitochondria. In vitro studies have shown that insulin can signal specifically via phosphatidylinositol-3-OH-kinase (PI3-K) and Akt to prevent cytochrome c release. Therefore, insulin may exert its neuroprotective effects during brain reperfusion by blocking cytochrome c release. We hypothesized that insulin acts through PI3-K, Akt, and Bcl-2 family proteins to inhibit cytochrome c release following transient global brain ischemia. We found that a single bolus of insulin given immediately upon reperfusion inhibited cytochrome c release for at least 24 h, and produced a fivefold improvement in neuronal survival at 14 days. Moreover, insulin’s ability to inhibit cytochrome c release was completely dependent on PI3-K signaling and insulin induces phosphorylation of Akt through PI3-K. In untreated animals, there was an increase in mitochondrial Bax at 6 h of reperfusion, and Bax binding to Bcl-XL was disrupted at the mitochondria. Insulin prevented both these events in a PI3-K-dependent manner. In summary, insulin regulates cytochrome c release through PI3-K likely by activating Akt, promoting the binding between Bax and Bcl-XL, and by preventing Bax translocation to the mitochondria. [ABSTRACT FROM AUTHOR]

This Perspective covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. Vacuum matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source without the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, inter alia , an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence. We, and others, have demonstrated exceptional analytical utility. Our current research is focused on how best to understand, improve, and use these novel ionization processes through dedicated platforms and source developments. These ionization processes convert volatile and nonvolatile compounds from solid or liquid matrixes into gas-phase ions for analysis by mass spectrometry using, e.g. , mass-selected fragmentation and ion mobility spectrometry to provide accurate, and sometimes improved, mass and drift time resolution. The combination of research and discoveries demonstrated multiple advantages of the new ionization processes and established the basis of the successes that lead to the Biemann Medal and this Perspective. How the new ionization processes relate to traditional ionization is also presented, as well as how these technologies can be utilized in tandem through instrument modification and implementation to increase coverage of complex materials through complementary strengths.

Cardiomyocytes and neurons are highly susceptible to ischemia-reperfusion injury; accordingly, considerable effort has been devoted to elucidating the cellular mechanisms responsible for ischemia-reperfusion-induced cell death and developing novel strategies to minimize ischemia-reperfusion injury. Maintenance of mitochondrial integrity is, without question, a critical determinant of cell fate. However, there is emerging evidence of a novel and intriguing extension to this paradigm: mitochondrial dynamics (that is, changes in mitochondrial morphology achieved by fission and fusion) may play an important but as-yet poorly understood role as a determinant of cell viability. Focusing on heart and brain, our aims in this review are to provide a synopsis of the molecular mechanisms of fission and fusion, summarize our current understanding of the complex relationships between mitochondrial dynamics and the pathogenesis of ischemia-reperfusion injury, and speculate on the possibility that targeted manipulation of mitochondrial dynamics may be exploited for the design of novel therapeutic strategies for cardio- and neuroprotection.