Dynamic Physiological Processes Research Articles

O-GlcNAc Transferase Enables AgRP Neurons to Suppress Browning of White Fat

Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here, we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting.

Information Processing in Computer-Assisted Interventions: 4th International Conference, 2013.

Information Processing in Computer-Assisted Interventions: 4th International Conference, 2013.

Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility

Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that, in the steady state, muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven crosstalk between muscularis macrophages and enteric neurons that controls gastrointestinal motility. PAPERFLICK:

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Corrigendum

Journal of NeurochemistryVolume 128, Issue 2 p. 340-340 CorrigendumFree Access Corrigendum This article corrects the following: Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission ChiHye Chung, Jesica Raingo, Volume 126Issue 2Journal of Neurochemistry pages: 146-154 First Published online: April 23, 2013 First published: 10 December 2013 https://doi.org/10.1111/jnc.12631AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Barrantes (2013), citation and reference details to the articles mentioned within the Preface were omitted from the published version of the paper. The corrected paragraphs and complete reference list are provided below: Synaptic vesicle dynamics and (trans-)synaptic regulation, co-authored by the Company of Biologists Young Investigator Awardee Jesica Raingo, of the University of La Plata, and ChiHye Chung, Konkuk University, describes various facets of pre-synaptic vesicle dynamics and trans-synaptic regulation (Chung and Raingo 2013). Tomoaki Shirao, Neurobiology & Behavior, Gunma University, and Christian González-Billault, University of Santiago, summarize cytoskeletal regulation of dendritic spine formation (Shirao and González-Billault 2013). Nanoscopic imaging (STED microscopy) developed by Prof. Stefan Hell's group at the Max-Planck Institute for Biophysical Chemistry in Göttingen is summarized by Christian Eggeling, Katrin Willig, and Francisco Barrantes (Eggeling et al. 2013). STED microscopy enables subdiffractional imaging of membrane dynamics, synapses or dendritic spines, opening new avenues and unique opportunities for future structural and functional analysis of these important structures. A related methodological review was contributed by Daniel Gitler, Kevin Staras, and Dan Mikulincer on Fluorescence Recovery after Photobleaching (FRAP) (Staras et al. 2013). The pathology of the synapse is also analyzed in various reviews. Jean-Nöel Octave, Catholic University of Louvain, Journal of Neurochemistry's former Editor-in-Chief Tony Turner, University of Leeds, and their co-authors describe the implications of APP in Alzheimer disease (Octave et al. 2013). Development and maintenance of the synapse and implications for psychiatric disorders are reviewed by Peter Penzes, Northwestern University, Andrés Buonanno, NIH in Bethesda, and Carlo Sala from the CNR Institute of Neuroscience in Milan (Penzes et al. 2013). All in all, the mini-reviews span considerable width and depth of cutting-edge subjects in current synaptic biology and synaptic diseases, the so-called synaptopathies. References Chung C. and Raingo J. (2013) Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission. J. Neurochem. 126, 146– 154. Eggeling C., Willig K. I. and Barrantes F. J. (2013) STED microscopy of living cells – new frontiers in membrane and neurobiology. J. Neurochem. 126, 203– 212. Octave J.-N., Pierrot N., Santos S. F., Nalivaeva N. N. and Turner A. J. (2013) From synaptic spines to nuclear signaling: nuclear and synaptic actions of the amyloid precursor protein. J. Neurochem. 126, 183– 190. Penzes P., Buonanno A., Passafaro M., Sala C. and Sweet R. A. (2013) Developmental vulnerability of synapses and circuits associated with neuropsychiatric disorders. J. Neurochem. 126, 165– 182. Shirao T. and González-Billault C. (2013) Actin filaments and microtubules in dendritic spines. J. Neurochem. 126, 155– 164. Staras K., Mikulincer D. and Gitler D. (2013) Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching. J. Neurochem. 126, 213– 222. Volume128, Issue2January 2014Pages 340-340 ReferencesRelatedInformation

Time-Varying Modeling of Cerebral Hemodynamics

The scientific and clinical importance of cerebral hemodynamics has generated considerable interest in their quantitative understanding via computational modeling. In particular, two aspects of cerebral hemodynamics, cerebral flow autoregulation (CFA) and CO2 vasomotor reactivity (CVR), have attracted much attention because they are implicated in many important clinical conditions and pathologies (orthostatic intolerance, syncope, hypertension, stroke, vascular dementia, mild cognitive impairment, Alzheimer's disease, and other neurodegenerative diseases with cerebrovascular components). Both CFA and CVR are dynamic physiological processes by which cerebral blood flow is regulated in response to fluctuations in cerebral perfusion pressure and blood CO2 tension. Several modeling studies to date have analyzed beat-to-beat hemodynamic data in order to advance our quantitative understanding of CFA-CVR dynamics. A confounding factor in these studies is the fact that the dynamics of the CFA-CVR processes appear to vary with time (i.e., changes in cerebrovascular characteristics) due to neural, endocrine, and metabolic effects. This paper seeks to address this issue by tracking the changes in linear time-invariant models obtained from short successive segments of data from ten healthy human subjects. The results suggest that systemic variations exist but have stationary statistics and, therefore, the use of time-invariant modeling yields "time-averaged models" of physiological and clinical utility.

Real-Time Electrical Impedimetric Monitoring of Blood Coagulation Process under Temperature and Hematocrit Variations Conducted in a Microfluidic Chip

Blood coagulation is an extremely complicated and dynamic physiological process. Monitoring of blood coagulation is essential to predict the risk of hemorrhage and thrombosis during cardiac surgical procedures. In this study, a high throughput microfluidic chip has been developed for the investigation of the blood coagulation process under temperature and hematocrit variations. Electrical impedance of the whole blood was continuously recorded by on-chip electrodes in contact with the blood sample during coagulation. Analysis of the impedance change of the blood was conducted to investigate the characteristics of blood coagulation process and the starting time of blood coagulation was defined. The study of blood coagulation time under temperature and hematocrit variations was shown a good agreement with results in the previous clinical reports. The electrical impedance measurement for the definition of blood coagulation process provides a fast and easy measurement technique. The microfluidic chip was shown to be a sensitive and promising device for monitoring blood coagulation process even in a variety of conditions. It is found valuable for the development of point-of-care coagulation testing devices that utilizes whole blood sample in microliter quantity.

Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi-quantitative approaches to their interpretation.

4-D Photoacoustic Tomography

Photoacoustic tomography (PAT) offers three-dimensional (3D) structural and functional imaging of living biological tissue with label-free, optical absorption contrast. These attributes lend PAT imaging to a wide variety of applications in clinical medicine and preclinical research. Despite advances in live animal imaging with PAT, there is still a need for 3D imaging at centimeter depths in real-time. We report the development of four dimensional (4D) PAT, which integrates time resolutions with 3D spatial resolution, obtained using spherical arrays of ultrasonic detectors. The 4D PAT technique generates motion pictures of imaged tissue, enabling real time tracking of dynamic physiological and pathological processes at hundred micrometer-millisecond resolutions. The 4D PAT technique is used here to image needle-based drug delivery and pharmacokinetics. We also use this technique to monitor 1) fast hemodynamic changes during inter-ictal epileptic seizures and 2) temperature variations during tumor thermal therapy.

Ontology of physics for biology: representing physical dependencies as a basis for biological processes

BackgroundIn prior work, we presented the Ontology of Physics for Biology (OPB) as a computational ontology for use in the annotation and representations of biophysical knowledge encoded in repositories of physics-based biosimulation models. We introduced OPB:Physical entity and OPB:Physical property classes that extend available spatiotemporal representations of physical entities and processes to explicitly represent the thermodynamics and dynamics of physiological processes. Our utilitarian, long-term aim is to develop computational tools for creating and querying formalized physiological knowledge for use by multiscale “physiome” projects such as the EU’s Virtual Physiological Human (VPH) and NIH’s Virtual Physiological Rat (VPR).ResultsHere we describe the OPB:Physical dependency taxonomy of classes that represent of the laws of classical physics that are the “rules” by which physical properties of physical entities change during occurrences of physical processes. For example, the fluid analog of Ohm’s law (as for electric currents) is used to describe how a blood flow rate depends on a blood pressure gradient. Hooke’s law (as in elastic deformations of springs) is used to describe how an increase in vascular volume increases blood pressure. We classify such dependencies according to the flow, transformation, and storage of thermodynamic energy that occurs during processes governed by the dependencies.ConclusionsWe have developed the OPB and annotation methods to represent the meaning—the biophysical semantics—of the mathematical statements of physiological analysis and the biophysical content of models and datasets. Here we describe and discuss our approach to an ontological representation of physical laws (as dependencies) and properties as encoded for the mathematical analysis of biophysical processes.

Development of a Continuous Dissolution/Absorption System—a Technical Note

It has been several decades since the compendial dissolution technique has been used to characterize the performance of oral drug products and has attempted to serve as a surrogate for in vivo bioequivalence testing (1). Provided a validated in vitro–in vivo correlation (IVIVC) is established, the demonstrative bioequivalence test based on clinical trials could be waived. This regulatory benefit increases the interest in linking dissolution results with pharmacokinetic performance. However, the outcomes based on previous research indicate that only a small portion of drugs (in Biopharmaceutics Classification System (BCS) class I, high solubility, high permeability) are successful in establishing IVIVCs and subsequently obtaining waives for bioequivalence tests (2). The key parameters controlling oral drug absorption are the solubility/dissolution of the drug in the gastrointestinal (GI) track and the permeability of the drug through the GI membrane. The current compendial methods are simplified models to the human GI tract and are limited in their abilities to simulate the dynamic aspects of the in vivo processes that are associated with dosage form transit through a complex GI environment. Compartmental absorption and transit models have been developed for years (3,4). Some progress has been made to develop a dissolution apparatus based on multicompartmental models such as: (1) integrate dissolution testing with in vitro permeation process or (2) simulate drug dynamic transit in the GI tract. James Polli's group developed an integrated dissolution/Caco-2 system, which could be used to predict dissolution–absorption relationships, as well as the contributions of dissolution and intestinal permeation to overall drug absorption (2,5). The system has been applied to study piroxicam, metoprolol, and ranitidine formulations, and the results were in agreement with clinical studies. Some similar reports were also published recently by Makoto Kataoka's group on a novel dissolution/permeation (D/P) system (6–8). The D/P system included an apical side and a basal side with a Caco-2 monolayer in between. It was reported that drug dissolution and permeation could be simultaneously determined with this system. Another approach to assess bioavailability/bioequivalence in vitro is the TIM-1 system developed at TNO Nutrition and Food Research, Netherlands (9). This system is a multicompartmental, dynamic, computer-controlled system that closely simulates in vivo dynamic physiological processes in the human upper GI tract. Those efforts make it possible to better simulate the GI tract and correlate in vitro drug performance to in vivo clinical trials. However, because of the complexity of these systems, some limitations such as result reproducibility and repeatability, cost of each test, ease of use of the method, and high throughput may prevent the wide use of these systems. A new dissolution/absorption system that takes the advantage of current progress and improves the reproducibility and repeatability of test results, at a low cost, would be of benefit. This article is focused on the development of a new dissolution/absorption system. The system still depends on the idea of multicompartmental simulation and is able to link drug dissolution and absorption performance. On the other hand, the new system has some improvements: (1) it employs a dissolution section and an absorption section (two compartments) instead of using four compartments as in the TIM-1 system; (2) it applies an artificial membrane to link dissolution and absorption without using a Caco-2 monolayer; and (3) it accommodates current USP official dissolution apparatus easily into the system. Some preliminary tests with two drug products (Inderal propranolol capsule and Uricalm phenazopyridine tablet) have been done with this newly developed system. The results indicate that this new system is flexible and easy to use, has low cost and maintenance, and may have potential to better correlate in vitro testing with in vivo clinical trials than current dissolution configurations.

The effect of teucrium polium honey on the wound healing and tensile strength in rat.

Wound healing represents a dynamic physiological process influenced by many factors. The aim of the present study was to evaluate the effects of Teucrium polium honey on the wound healing and tensile strength in rat. Thirty-six Sprague-Dawley rats were randomly divided into four equal (n= 9) treatment and control groups. Two full-thickness wounds were made over the dorsal thoracic region according to the incision and excision models. Animals were treated with topical Teucrium polium honey twice a day post surgery until complete healing was achieved. Histopathology and tensiometry were then studied. The wound healing process occurred faster in the incision model than excision ones (P< 0.05). Teucrium polium honey promoted wound contraction, closure time and tensile strength (P< 0.05). Histopathological study also showed relative epithelial proliferation, improved angiogenesis granulation, and fibrous connective tissue in Teucrium polium honey treated animals. The present study demonstrates that Teucrium polium honey can accelerate wound healing as well as tensile strength in rat skin wounds.

Real-Time Hyperspectral Imaging of Multiple Biosensors in Pancreatic Beta Cells

Spectral imaging techniques are important for many biological experiments, particularly live-cell quantitative imaging of multiple fluorescence probes. Existing hyperspectral imaging systems require sequential techniques, limiting the data acquisition rate. A newly-developed snapshot device, the Image Mapping Spectrometer (IMS), acquires full spectral information simultaneously from every pixel in the field with image acquisition rates up to 10 frames/second. The IMS maps adjacent pixels from the object to create space between them in the image, and then uses a grating to spread wavelength content from each pixel into this space. Direct image re-mapping provides the final 3D (x, y, lambda) data cube. Fluorescent protein (FP)-based biosensors are increasingly valuable tools for identifying subcellular dynamic processes in live cells. Many biosensors are based on FP-FRET, and measurements of the resultant small changes in FRET require high quality data. Tracking intracellular free Ca2+ levels is also crucial to elucidating signaling events, but the best Ca2+ indicator dyes overlap in the spectral emission range of the common FP-FRET biosensors. We have used the IMS system to simultaneously image multicolored FPs (i.e., CFP, GFP, YFP) in combination with extrinsic indicator dyes, such as Fluo-4. To demonstrate the speed and resolution of the IMS approach, we monitored both intracellular Ca2+ oscillations and caspase-3 activity during hydrogen peroxide-induced apoptosis. Ca2+ activity was measured with Fluo-4 (emission peak at 517 nm) and caspase-3 activity was measured with SCAT3.1, a FRET biosensor based on the ECFP (emission peak at 484 nm) and EYFP (emission peak at 527 nm) pair. Using the IMS, the three fluorophores were imaged with sub-second temporal resolution and spectrally unmixed in real-time. This permits direct correlation of Ca2+ activity with other apoptotic signaling events and demonstrates the power of the IMS for measuring dynamic physiological processes.

Development of a simple model device for in vitro gastric digestion investigation

There have been some reports in the literature of model gastric digestion systems to mimic the dynamic physiological processes within the gastrointestinal tract. However, such devices often require the specification of many control parameters making routine digestion tests unfeasible. This paper introduces a simple in vitro digestion device, comprising of a water-jacketed glass vessel into which a spherical Teflon probe of variable diameter can be inserted. The probe is controlled by a texture analyser to simulate the kinetics of a food digestion process. Using this device under well controlled hydrodynamic flow and biochemical conditions key digestion parameters such as pH, food particle size, protein release, lipid release, cloudiness, etc, can be determined. Feasibility tests of the model device have been conducted using roasted and non-roasted peanuts particles. The status of peanut digestion was examined by the changes in particle size distribution and the mean particle size. Significant differences of surface microstructure have also been observed for peanut particles after the digestion. The influence of parameters such as food to gastric juice ratio, the probe speed and pepsin concentration have been examined in this work. Initial results confirm that all these factors influence the kinetic process of gastric digestion considerably and should be well regulated in any in vitro digestion investigations. We propose that the model device has the advantages of easy control and operation and furthermore could be an ideal tool for routine in vitro gastric digestion studies.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes.

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R(2) = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R(2) = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being.

Dynamic changes to survivin subcellular localization are initiated by DNA damage

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian's subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality.

A Parallel Excitation Based Fluorescence Molecular Tomography System for Whole-Body Simultaneous Imaging of Small Animals

Challenges remain in imaging complete dynamic physiological processes in vivo through the whole small animal body using fluorescence molecular tomography (FMT). In this article, a novel non-contact full-angle FMT system that enables whole-body simultaneous imaging of small animals is presented. The whole-body simultaneous imaging ability is achieved by employing a line-shaped parallel excitation source, which can provide extended spatial sampling dataset to reconstruct multiple fluorescent targets distributed in whole animal body during one full-angle FMT imaging process. The key performances of this system were evaluated by a series of experiments. Quantitation linearity for over two orders of magnitude of fluorescence markers concentration was demonstrated, and an accessible simultaneous imaging domain of 4.0 x 1.5 cm² could be achieved utilizing the parallel excitation pattern. Moreover, the in vivo imaging feasibility and performance were validated by localizing two fluorescent targets implanted at different positions of a nude mouse. The results suggest that compared with conventional single point excitation FMT system, the proposed system can achieve a whole-body simultaneous imaging domain and impart the ability to image complete dynamic physiological processes in vivo.

Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2

Some isoforms of secretory phospholipase A(2) (sPLA(2)) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA(2) isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6-48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA(2) were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.

Physiological implications of natural versus induced arousal from torpor

Hibernation is a dynamic physiological process wherein animals cycle between periods of torpor and periods of interbout arousal. Periodically, animals spontaneously exit torpor and rewarm to euthermic Tb; this rewarming process is called arousal. Arousal from torpor is a rapid event. During arousal increases in oxygen consumption, Tb, and the restoration of blood flow occur asynchronously i.e. at different times and at different rates. In addition to natural arousals, animals can also be induced to arouse prematurely in response to mild disturbances. Historically, natural and induced arousals have been regarded as synonymous events. We question the verity of this assumption. To address this question, we have examined plasma metabolites and respiratory rate during natural and induced arousals. We are also examining markers of oxidative damage. Analysis of plasma metabolites has revealed a significant (p&lt;0.05) increase in lactate concentration during induced arousal whereas plasma glucose and free fatty acids levels are unchanged (p&gt;0.05). Preliminary respirometry measurements indicate that the first 30 minutes of induced arousal may be differentially fueled or involve changes in acid/base regulation. Brain, heart, liver, and kidney samples collected from arousing animals at body temperatures of 10°, 20°, and 30°C are being assayed for indications of oxidative damage to proteins, lipids, and DNA.