Modulation of respiration during brain hypoxia

This review is a summary of the effects of brain hypoxia on respiration with a particular emphasis on those studies relevant to understanding the cellular basis of these effects. Special attention is given to mechanisms that may be responsible for the respiratory depression that appears to be the primary sequela of brain hypoxia in animal models. Although a variety of potential mechanisms for hypoxic respiratory depression are considered, emphasis is placed on changes in the neuromodulator constituency of the respiratory neuron microenvironment during hypoxia as the primary cause of this phenomenon. Hypoxia is accompanied by a net increase in neuronal inhibition due to both decreased excitatory and increased inhibitory neuromodulator levels. A survey of hypoxia-tolerant cellular systems and organisms suggests that hypoxic respiratory depression may be a manifestation of the depression of cellular metabolism, which appears to be a major adaptation to limited oxygen availability in these systems.     

Active reversal of motor memories reveals rules governing memory encoding

Learning systems must be able to store memories reliably, yet be able to modify them when new learning is required. At the mechanistic level, new learning may either reverse the cellular events mediating the storage of old memories or mask the old memories with additional cellular changes that preserve the old cellular events in a latent form. Behavioral evidence about whether reversal or masking occurs in a particular circuit can constrain the cellular mechanisms used to store memories. Here we examine these constraints for a simple cerebellum-dependent learning task, motor learning in the vestibulo-ocular reflex (VOR). Learning can change the amplitude of the VOR in two opposite directions. Contrary to previous models about memory encoding by the cerebellum, our results indicate that these behavioral changes are implemented by different plasticity mechanisms, which reverse each other with unequal efficacy.     

Recent developments in the intracellular degradation of oxidized proteins

The accumulation of oxidized proteins in cells and tissues is a feature of a number of age-related diseases and may also occur as a result of the aging process itself. In this article we review recent advances in our understanding of the cellular degradation of oxidized proteins directing our attention primarily to information which directly bears on the behavior of intact eukaryotic cells. We summarize new work on the key intracellular degradative machineries, proteasomes and lysosomes and examine evidence implicating an increase in protein hydrophobicity as the primary signal to the proteasome to initiate degradation. The data identifying the proteasome as the main route of degradation of oxidized proteins is examined, as well as recent data investigating changes in proteasome function after exposure of cells to oxidants and the altered catabolism of oxidized proteins in aging cells. Evidence for the cooperation between the lysosomal and proteasomal systems in the degradation of oxidized proteins is discussed. We conclude that the cellular catabolism of oxidized proteins may be a more complex process than it first appeared and suggest key issues that need to be resolved to improve our understanding of this important process.     

The targeting of plant cellular systems by injected type III effector proteins

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector–host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.     

Genetic modifiers of Drosophila palmitoyl-protein thioesterase 1-induced degeneration

Infantile neuronal ceroid lipofuscinosis (INCL) is a pediatric neurodegenerative disease caused by mutations in the human CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase 1 (PPT1), suggesting an important role for the regulation of palmitoylation in normal neuronal function. To further elucidate Ppt1 function, we performed a gain-of-function modifier screen in Drosophila using a collection of enhancer-promoter transgenic lines to suppress or enhance the degeneration produced by overexpression of Ppt1 in the adult visual system. Modifier genes identified in our screen connect Ppt1 function to synaptic vesicle cycling, endo-lysosomal trafficking, synaptic development, and activity-dependent remodeling of the synapse. Furthermore, several homologs of the modifying genes are known to be regulated by palmitoylation in other systems and may be in vivo substrates for Ppt1. Our results complement recent work on mouse Ppt1(-/-) cells that shows a reduction in synaptic vesicle pools in primary neuronal cultures and defects in endosomal trafficking in human fibroblasts. The pathways and processes implicated by our modifier loci shed light on the normal cellular function of Ppt1. A greater understanding of Ppt1 function in these cellular processes will provide valuable insight into the molecular etiology of the neuronal dysfunction underlying the disease.     

Intracellular proteolytic systems may function as secondary antioxidant defenses: An hypothesis

In recent years it has become clear that various free radicals and related oxidants can cause serious damage to intracellular enzymes and other proteins. Several investigators have shown that in extreme cases this can result in an accumulation of oxidatively damaged proteins as useless cellular debris. In other instances, proteins may undergo scission reactions with certain radicals/oxidants, resulting in the direct formation of potentially toxic peptide fragments. Data has also been gathered (recently) demonstrating that various intracellular proteolytic enzymes or systems can recognize, and preferentially degrade, oxidatively damaged proteins (to amino acids). In this hypothesis paper I present evidence to suggest that proteolytic systems (of proteinases, proteases, and peptidases) may function to prevent the formation or accumulation of oxidatively damaged protein aggregates. Proteolytic systems can also preferentially degrade peptide fragments and may thus prevent a wide variety of potentially toxic consequences. I propose that many proteolytic enzymes may be important components of overall antioxidant defenses because they can act to ameliorate the consequences of oxidative damage. A modified terminology is suggested in which the primary antioxidants are such agents as vitamin E, β-carotene, and uric acid and such enzymes as Superoxide dismutase, glutathione peroxidase, and DT-diaphorase. In this classification scheme, proteolytic systems, DNA repair systems, and certain lipolytic enzymes would be considered as secondary antioxidant defenses. As secondary antioxidant defenses, proteolytic systems may be particularly important in times of high oxidative stress, during periods of (primary) antioxidant insufficiency, or with advancing age.     

Comparisons of tests for aneuploidy

The fundamental problems that face us in the development of suitable assay systems for the detection of potentially aneugenic (aneuploidy-inducing) chemicals include: (a) the diversity of cellular targets and mechanisms where perturbations of structure and function may give rise to changes in chromosome number, and (b) the phylogenetic differences that exist between species in their mechanism and kinetics of cell division and their metabolic profiles. A diverse range of assay systems have been developed, which have been shown to have potential for use in the detection of either changes in chromosome number or of perturbations of the events which may be causal in the induction of aneuploidy.

Chromosome number changes may be detected cytologically by karyotypic analysis, or by the use of specialised strains in which aneuploid progeny may be observed due to phenotypic differences with aneuploid parental cells or whole organisms. Techniques for the detection of cellular target modifications range from in vitro studies of tubulin polymerisation to observations of the behaviour of various cellular organelles and their fidelity of action during the division cycle.

The diversity of mechanisms which may give rise to aneuploidy and the qualitative relevance of events observed in experimental organisms compared to man make it unlikely that the detection and risk assessment of the aneugenic activity of chemicals will be possible using a single assay system. Optimal screening and assessment procedures will thus be dependent upon the selection of an appropriate battery of predictive tests for the measurement of the potentially damaging effects of aneuploidy induction.     

Is serum gamma glutamyltransferase a marker of oxidative stress?

The primary role of cellular gamma glutamyltransferase (GGT) is to metabolize extracellular reduced glutathione (GSH), allowing for precursor amino acids to be assimilated and reutilized for intracellular GSH synthesis. Paradoxically, recent experimental studies indicate that cellular GGT may also be involved in the generation of reactive oxygen species in the presence of iron or other transition metals. Although the relationship between cellular GGT and serum GGT is not known and serum GGT activity has been commonly used as a marker for excessive alcohol consumption or liver diseases, our series of epidemiological studies consistently suggest that serum GGT within its normal range might be an early and sensitive enzyme related to oxidative stress. For example, serum and dietary antioxidant vitamins had inverse, dose-response relations to serum GGT level within its normal range, whereas dietary heme iron was positively related to serum GGT level. More importantly, serum GGT level within its normal range positively predicted F2-isoprostanes, an oxidative damage product of arachidonic acid, and fibrinogen and C-reactive protein, markers of inflammation, which were measured 5 or 15 years later, in dose-response manners. These findings suggest that strong associations of serum GGT with many cardiovascular risk factors and/or events might be explained by a mechanism related to oxidative stress. Even though studies on serum and/or cellular GGT is at a beginning stage, our epidemiological findings suggest that serum GGT might be useful in studying oxidative stress-related issues in both epidemiological and clinical settings.     

Determination of cellular strains by combined atomic force microscopy and finite element modeling

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease.     

Integration of Metabolic Reactions and Gene Regulation

Metabolic reactions and gene regulation are two primary processes of cells. In response to environmental changes cells often adjust the regulatory programs and shift the metabolic states. An integrative investigation and modeling of these two processes would improve our understanding about the cellular systems and may generate substantial impacts in medicine, agriculture, environmental protection, and energy production. We review the studies of the various aspects of the crosstalk between metabolic reactions and gene regulation, including models, empirical evidence, and available databases.     

Why modules matter

The serendipitous discovery of the SH2 domain unleashed a sea-change in our conceptual molecular understanding of protein function. The reductionist approaches that followed from the recognition of modular protein interaction domains transformed our understanding of cellular signal transduction systems, how they evolve and how they may be manipulated. We now recognize thousands of conserved protein modules - many of which have been described in structure and function, implicated in disease, or underlie targeted therapeutics. The reductionist study of isolated protein modules has enabled the reconstruction of the protein interaction networks that underlie cellular signalling. Protein modules themselves are becoming tools to probe cellular activation states and identify key interactions hubs in both normal and diseased cells and the concept of protein modularity is central to the field of synthetic biology. This brief word of introduction serves to highlight the historical impact of the very powerful idea of protein modules and sets the stage for the exciting on-going discoveries discussed in this issue.     

The role of calcium in exocytosis and endocytosis in plant cells

The role of calcium in the individual cellular events leading to exocytosis is considered. Both vesicle movement processes and vesicle fusion at the cell surface require calcium for completion of specific events in this pathway. Our knowledge of these events is incomplete. In particular the movement of secretory vesicles by the cytoskeleton in response to added calcium is a key event that is beyond our comprehension at present. At the whole cell level, it is shown that external calcium, at the appropriate concentration, is required to elicit secretion at optimal rates. In both plant and animal cells secretion appears to be dependent on, or is triggered by, a rise in the level of internal free calcium ions from about 10^-7 to 10^-6M or even higher. In these eukaryotes internal organelles take up calcium and maintain a low level of calcium in the cell, offsetting the inflow of calcium from the plasma membrane. In some systems the inflow is restricted to a certain part of the plasma membrane, which then acts as a focus for exocytosis and, thereby, establishes a cellular polarity. In plant tissues there appears to be a requirement for some circulation of calcium within the apoplast, to sustain secretion. Recent papers on endocytosis have confirmed its occurrence in plant cells and made significant advances in isolating and characterising the clathrin coats of the coated vesicles involved in the uptake. There is no evidence, at present, for a direct role for calcium in these events. Indirectly, calcium stimulates exocytosis, and hence the delivery of excess membrane to the cell surface, which may be retrieved by an increase in the rate of endocytosis. Quantitative comparisons of the membrane flow occurring in these pathways are not available. Several plant cellular systems have been employed to study secretion and some of these may prove to be superior model systems for the investigation of certain aspects of the control of exocytosis and endocytosis by calcium ions.     

Enzymatic studies of DNA repair in Drosophila melanogaster

Thus far, our studies in Drosophila have concentrated primarily on the various enzymes involved in the in vitro repair of modified or nonconventional DNA substrates. In some cases, our findings have led us to investigate events that may not have a bearing on DNA repair, but rather may be associated with developmental signals important to the maturation of the organism. As appealing as some of these models seem, however, they must await confirmation through detailed genetic studies before any substantial conclusions can be drawn. This combination of genetic and biochemical knowledge makes Drosophila an exciting organism for an eventual detailed understanding of the developmental expression and cellular location of DNA-repair systems.     

Electrophysiological effects of MCH on neurons in the hypothalamus

Melanin concentrating hormone (MCH) has been implicated in many brain functions and behaviors essential to the survival of animals. The hypothalamus is one of the primary targets where MCH-containing nerve fibers and MCH receptors are extensively expressed and its actions in the brain are exerted. Since the identification of MCH receptors as orphan G protein coupled receptors, the cellular effects of MCH have been revealed in many non-neuronal expression systems (including Xenopus oocytes and cell lines), however, the mechanism by which MCH modulates the activity in the neuronal circuitry of the brain is still under investigation. This review summarizes our current knowledge of electrophysiological effects of MCH on neurons in the hypothalamus, particularly in the lateral hypothalamus. Generally, MCH exerts inhibitory effects on neurons in this structure and may serve as a homeostatic regulator in the lateral hypothalamic area. Given the contrast between the limited data on cellular functions of MCH in the hypothalamus versus a fast growing body of evidence on the vital role of MCH in animal behavior, further investigations of the former are warranted.