Modification of Proteins by Metabolites in Immunity.

Immunometabolism has emerged as a key focus for immunologists, with metabolic change in immune cells becoming as important a determinant for specific immune effector responses as discrete signaling pathways. A key output for these changes involves post-translational modification (PTM) of proteins by metabolites. Products of glycolysis and Krebs cycle pathways can mediate these events, as can lipids, amino acids, and polyamines. A rich and diverse set of PTMs in macrophages and T cells has been uncovered, altering phenotype and modulating immunity and inflammation in different contexts. We review the recent findings in this area and speculate whether they could be of use in the effort to develop therapeutics for immune-related diseases.

Short-term effects of military fog oil on the fountain darter (Etheostoma fonticola).

Toxicity tests evaluated chronic and sublethal effects of fog oil (FO) on a freshwater endangered fish. FO is released during military training as an obscurant smoke that can drift into aquatic habitats. Fountain darters, Etheostoma fonticola, of four distinct life stages were exposed under laboratory conditions to three forms of FO. FO was vaporized into smoke and allowed to settle onto water, violently agitated with water, and dosed onto water followed by photo-oxidization by ultraviolet irradiation. Single smoke exposures of spawning adult fish did not affect egg production, egg viability, or adult fish survival in 21-day tests. Multiple daily smoke exposures induced mortality after 5 days for larvae fish. Larvae and juvenile fish were more sensitive than eggs in 96-h lethal concentration (LC50) tests with FO­water mixtures and photo-oxidized FO. Water-soluble FO components photo-modified by ultraviolet radiation were the most toxic, thus indicating the value of examining weathering and aging of chemicals for the best determination of environmental impact.

Single-vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode.

The nature of synaptic vesicle recycling at nerve terminals has been a subject of considerable debate for >35 years. Here, we report the use of an optical strategy that allows the exocytosis and retrieval of synaptic components to be tracked in real time at single-molecule sensitivity in living nerve terminals. This approach has allowed us to examine the recycling of synaptic vesicles in response to single action potentials. Our results show that, after exocytosis, individual synaptic vesicles are retrieved by a stochastic process with an exponential distribution of delay times, with a mean time of approximately 14 s. We propose that evidence for fast endocytosis, such as that proposed to support the presence of kiss-and-run, is likely explained by the stochastic nature of a slower process.

NCAM and vesicle cycling: the importance of good glue in the long run.

Careful functional dissection of mouse neuromuscular junctions (NMJs) formed in the absence of NCAM reveal that in spite of relatively normal morphology and ultrastructure, the presynaptic terminals have profound alterations in the mechanisms of recycling of synaptic vesicles. These alterations, including the appearance of a brefeldin-sensitive pathway, leave synaptic transmission impaired for certain types of activity. NCAMs thus appear to play a critical role in the molecular organization and function of synaptic terminals.

Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion.

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Synapsin dispersion and reclustering during synaptic activity.

Presynaptic modulation of synaptic transmission provides an important basis for control of synaptic function. The synapsins, a family of highly conserved proteins associated with synaptic vesicles, have long been implicated in the regulation of neurotransmitter release. However, direct physiological measurements of the molecular mechanisms have been lacking. Here we show that in living hippocampal terminals, green fluorescent protein (GFP)-labeled synapsin Ia dissociates from synaptic vesicles, disperses into axons during action potential (AP) firing, and reclusters to synapses after the cessation of synaptic activity. Using various mutated forms of synapsin Ia that prevent phosphorylation at specific sites, we performed simultaneous FM 4-64 measurements of vesicle pool mobilization along with synapsin dispersion kinetics. These studies indicate that the rate of synapsin dispersion is controlled by phosphorylation, which in turn controls the kinetics of vesicle pool turnover. Thus synapsin acts as a phosphorylation-state-dependent regulator of synaptic vesicle mobilization, and hence, neurotransmitter release.

Presynaptic imaging techniques.

Understanding the detailed molecular events that support chemical synaptic transmission requires high-resolution methods that provide quantitative information combined with molecular specificity. In recent years, many new technological approaches, including genetically encoded fluorescent indicators, ultra-thin sectioning, and live-cell imaging have been brought to bear on understanding the cell biology and physiology of presynaptic terminals.

Measurements of vesicle recycling in central neurons.

Neurotransmitter-containing vesicles in presynaptic nerve terminals are essential for synaptic transmission. The vesicles undergo a cycle that leads to transmitter release by exocytosis followed by endocytosis and refilling of the vesicles. Here we discuss new optical methods that have helped researchers study this cycle at functional and molecular levels, which is essential for our understanding of the regulation of synaptic transmission.

Calcium accelerates endocytosis of vSNAREs at hippocampal synapses.

A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.

The use of pHluorins for optical measurements of presynaptic activity.

Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.

Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.

Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.

Long-term survival and functional capacity in cardiac surgery patients after prolonged intensive care.

OBJECTIVE: To determine whether hospital discharge alone represents a good outcome for patients who had prolonged intensive care after cardiac surgery by studying their postdischarge survival and functional outcome. The secondary objective is to estimate the proportion of intensive care unit (ICU) resources used by the long-stay (> or = 10 initial consecutive ICU days) patients and to identify preoperative patient characteristics that are associated with a prolonged ICU stay and hospital and long-term survival. DESIGN: Inception cohort study. SETTING: The Cleveland Clinic Foundation, a tertiary care, academic teaching institution. PATIENTS: Cardiac surgery patients with an initial ICU stay of 10 or more consecutive days. INTERVENTIONS: Data were collected daily during hospitalization on every adult who underwent coronary artery bypass graft and/or valve surgery at one institution in 1993. Discharged patients who spent >10 initial consecutive days in the ICU after surgery were contacted by telephone to determine vital status and functional capacity using the Duke Activity Status Index. Total ICU and total hospital direct costs were obtained for each patient. MEASUREMENTS AND MAIN RESULTS: The primary outcome measurements were ICU length of stay, hospital mortality, after-surgery and postdischarge mortality and functional capacity, and relative resource utilization. Of the 2,618 cardiac surgery patients who met the inclusion criteria, 142 (5.4%) had an initial ICU length of stay of 10 or more consecutive days. Of these, 47 (33.1%) died in the hospital. Ninety-four of the 95 discharged patients were followed up (median follow-up, 30.6 months), and 44 of the 94 (46.8%) died during the follow-up period. The median Duke Activity Status Index for the 50 survivors was 26 out of a possible 58.2. The 142 long-stay patients used 50% of the total ICU days and 48% of the total ICU direct cost for all 2,618 patients. CONCLUSIONS: Many survivors of prolonged intensive care die soon after hospital discharge and many longer term survivors have a poor functional state. Therefore, hospital discharge is an incomplete measure of outcome for these patients, and longer follow-up is more appropriate. The relatively small number of patients who require prolonged intensive care consumes a disproportionate amount of the total ICU and total hospital direct cost.

Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing.

During repetitive action potential firing the maintenance of synaptic transmission relies on a continued supply of synaptic vesicles for fusion with the presynaptic plasma membrane. The mechanism of transport by which vesicles are delivered to the site of fusion from a reserve pool is unknown, as are the biochemical pathways linking intracellular Ca2+ elevation with vesicle mobilization. Here, using the fluorescent tracer FM1-43 in hippocampal synaptic terminals, I show that inhibitors of myosin light chain kinase can block mobilization of the reserve pool and not the immediately releasable pool.

Predictors of outcome in cardiac surgical patients with prolonged intensive care stay.

OBJECTIVE: To determine the predictors of outcome in cardiac surgical patients with prolonged ICU stay. DESIGN: Inception cohort with retrospective chart review. SETTING: Adult cardiovascular ICU. PATIENTS: All patients admitted after cardiac surgery who stayed in ICU for at least 14 consecutive days. INTERVENTIONS: Collection of data, including preoperative demographics, comorbidity, routine laboratory testing, surgical procedure, duration of cardiopulmonary bypass and aortic cross-clamping, postoperative requirement for transfusion and intra-aortic balloon counterpulsation, and postoperative indexes of organ dysfunction 14 and 28 days after surgery. An organ failure score (OFS) was calculated for days 1, 14, and 28. OUTCOME MEASURES: Hospital mortality. RESULTS: One hundred forty-one of 324 (43.5%) ICU admissions lasting at least 14 days resulted in hospital mortality. Seventy-four of 166 (45%) ICU admissions lasting at least 28 days resulted in hospital mortality. Preoperative demographics, morbidity, and indexes of organ failure in the first 24 h after surgery were not predictive of hospital mortality. Indexes of organ failure predictive of hospital death at 14 days included requirement for epinephrine infusion, diminished Glasgow coma scale, requirement for dialysis, greater value of BUN, lower value of creatinine, greater value of bilirubin, greater value of arterial PCO2, lower platelet count, and lower value of serum albumin. After a 28-day stay in ICU, the indexes of organ failure predictive of hospital mortality included requirement for dopamine or norepinephrine infusions, diminished Glasgow coma score, greater value of bilirubin, greater value of arterial PCO2, lower value of serum albumin, and advanced age. The area under the receiver operating characteristic curve for the OFS on day 1 was 0.55+/-0.04 (p=0.12), on day 14 it was 0.75+/-0.03 (p<0.0001), and on day 28 it was 0.76+/-0.04 (p<0.0001). CONCLUSION: Preoperative health status and early organ failure were not predictive of late hospital mortality. The pattern of late organ failure associated with hospital mortality changed with time.

Optical detection of a quantal presynaptic membrane turnover.

Exploration of the mechanisms and plasticity of synaptic transmission has been hindered by the lack of a method to measure single vesicle turnover directly in individual presynaptic boutons at isolated nerve terminals. Although postsynaptic electrical recordings have provided a wealth of invaluable basic information about quantal presynaptic processes, this approach has often proved difficult to apply at most central nervous system synapses. Here we describe the direct optical detection of single quantal events in individual presynaptic boutons of cultured hippocampal neurons. Using the fluorescent dye FM 1-43 as a tracer for presynaptic endocytosis, we have characterized both evoked and spontaneous components of presynaptic function at the level of individual quanta. Our results are consistent with quantal interpretations of previous electrophysiological analyses and provide new information about the unitary membrane recycling event and its coupling to individual action potential stimuli, about spontaneous vesicle turnover at individual boutons, and about the numbers of vesicles recycling at individual boutons.

Synaptic vesicle recycling in synapsin I knock-out mice.

The synapsins are a family of four neuron-specific phosphoproteins that have been implicated in the regulation of neurotransmitter release. Nevertheless, knock-out mice lacking synapsin Ia and Ib, family members that are major substrates for cAMP and Ca2+/ Calmodulin (CaM)-dependent protein kinases, show limited phenotypic changes when analyzed electrophysiologically (Rosahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1995. Nature (Lond.). 375: 488-493; Rosahl, T.W., M. Geppert, D. Spillane, D., J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Sudhof. 1993. Cell. 75:661-670; Li, L., L.S. Chin, O. Shupliakov, L. Brodin, T.S. Sihra, O. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen. 1995. Proc. Natl. Acad. Sci. USA. 92:9235-9239; see also Pieribone, V.A., O. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard. 1995. Nature (Lond.). 375:493-497). Here, using the optical tracer FM 1-43, we characterize the details of synaptic vesicle recycling at individual synaptic boutons in hippocampal cell cultures derived from mice lacking synapsin I or wild-type equivalents. These studies show that both the number of vesicles exocytosed during brief action potential trains and the total recycling vesicle pool are significantly reduced in the synapsin I-deficient mice, while the kinetics of endocytosis and synaptic vesicle repriming appear normal.

Potentiation of evoked vesicle turnover at individually resolved synaptic boutons.

We have studied synaptic plasticity in hippocampal cell cultures using a new imaging approach that allows unambiguous discrimination of presynaptic function at the level of single synaptic boutons. Employing a protocol designed to test for use-dependent plasticity resembling N-methyl-D-aspartate receptor-dependent long-term potentiation (NMDA-type LTP), we find that brief tetanic stimuli induce a potentiation of evoked synaptic vesicle turnover that lasts for at least 1 hr. Induction of this clearly presynaptic potentiation is blocked by putative postsynaptic glutamate receptor antagonists, suggesting that a retrograde induction signal might be involved. Potentiation appears to occur approximately equally at boutons of low and high initial release probabilities, and evidently does not involve an increase in the size of the total recycling synaptic vesicle pool.