Glycolytic oscillations and intracellular K+ concentration are strongly coupled in the yeast Saccharomyces cerevisiae.

We measured temporal oscillations of intracellular K+ concentration in yeast cells exhibiting glycolytic oscillations using fluorescence spectroscopy and microscopy methods. These oscillations showed the same period as those of glycolytic metabolites (NADH, ATP), indicating a strong coupling between them. We experimentally ruled out that oscillations originate in extra- or intracellular K+ fluxes and conclude that these oscillations arise from fluctuations in free and adsorbed states of K+ in the cell interior. Oscillations in K+ showed a strong dependence on ATP and the organization of the cell cytoskeleton. Our results challenge the widely held view that intracellular K+ predominantly exists in a free state. They can, however, be productively understood in terms of Gilbert Ling's Association-Induction hypothesis.

Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach.

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

The innards of the cell: studies of water dipolar relaxation using the ACDAN fluorescent probe.

This article reviews the use of the 6-acetyl-2-(dimethylamino)naphthalene (ACDAN) fluorophore to study dipolar relaxation in cells, tissues, and biomimetic systems. As the most hydrophilic member of the 6-acyl-2-(dimethylamino)naphthalene series, ACDAN markedly partitions to aqueous environments. In contrast to 6-lauroyl-2-(dimethylamino)naphthalene (LAURDAN), the hydrophobic and best-known member of the series used to explore relaxation phenomena in biological (or biomimetic) membranes, ACDAN allows mapping of spatial and temporal water dipolar relaxation in cytosolic and intra-organelle environments of the cell. This is also true for the 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) derivative which, unlike LAURDAN, partitions to both hydrophobic and aqueous environments. We will (i) summarize the mechanism which underlies the solvatochromic properties of the DAN probes, (ii) expound on the importance of water relaxation to understand the intracellular environment, (iii) discuss technical aspects of the use of ACDAN in eukaryotic cells and some specialized structures, including liquid condensates arising from processes leading to liquid immiscibility and, (iv) present some novel studies in plant cells and tissues which demonstrate the kinds of information that can be uncovered using this approach to study dipolar relaxation in living systems.

Cellular metabolism and colloids: Realistically linking physiology and biological physical chemistry.

Important concepts from colloidal physical chemistry such as coacervation, phase transitions, emergent properties and ionic association, are currently emerging in the lexicon of cellular biology, prompted mostly by recent experimental observations of liquid phase coexistence in the cell cytosol. Nevertheless, from an historical point of view, the application of these concepts in cell biology is not new. They were key concepts into the so-called protoplasmic doctrine, an alternative (and largely forgotten) approach to cell physiology. The most complete theory originating from this line of thinking was the Association-Induction Hypothesis (AIH), introduced by Gilbert N. Ling in 1962. The AIH, which envisions living cells as complex dynamical colloidal systems, provides ample theory and experimental evidence to call into question the now dominant view of living cells as fluid-filled vesicles. This review attempts to present and discuss the usefulness of the AIH to understand a series of experimental observations from our laboratory from living suspensions of the yeast Saccharomyces cerevisiae exhibiting glycolytic oscillations. Particularly, the AIH helped us integrate, in a mechanistic sense, the basis of a strong temporal coupling observed between ATP and a series of cellular properties such as intracellular water dipolar relaxation, intracellular K+ concentration, among many others, where the colloidal physical chemistry of the cell interior plays a fundamental role.

Lipids, membranes, colloids and cells: A long view.

This paper revisits long-standing ideas about biological membranes in the context of an equally long-standing, but hitherto largely unappreciated, perspective of the cell based on concepts derived from the physics and chemistry of colloids. Specifically, we discuss important biophysical aspects of lipid supramolecular structure to understand how the intracellular milieu may constrain lipid self-assembly. To this end we will develop four lines of thought: first, we will look at the historical development of the current view of cellular structure and physiology, considering also the plurality of approaches that influenced its formative period. Second, we will review recent basic research on the structural and dynamical properties of lipid aggregates as well as the role of phase transitions in biophysical chemistry and cell biology. Third, we will present a general overview of contemporary studies into cellular compartmentalization in the context of a very rich and mostly forgotten general theory of cell physiology called the Association-Induction Hypothesis, which was developed around the time that the current view of cells congealed into its present form. Fourth, we will examine some recent developments in cellular studies, mostly from our laboratory, that raise interesting issues about the dynamical aspects of cell structure and compartmentalization. We will conclude by suggesting what we consider are relevant questions about the nature of cellular processes as emergent phenomena.

Bringing antivenoms to Sub-Saharan Africa.

To reduce unacceptably high death rates from snakebite envenomation, sub-Saharan Africa must adopt not only a new generation of multivalent biotech antivenoms, but also an infrastructure to deliver them.

Inductive effects in amino acids and peptides: Ionization constants and tryptophan fluorescence.

Although inductive effects in organic compounds are known to influence chemical properties such as ionization constants, their specific contribution to the properties/behavior of amino acids and functional groups in peptides remains largely unexplored. In this study we developed a computationally economical algorithm for ab initio calculation of the magnitude of inductive effects for non-aromatic molecules. The value obtained by the algorithm is called the Inductive Index and we observed a high correlation (R2 = 0.9427) between our calculations and the pKa values of the alpha-amino groups of amino acids with non-aromatic side-chains. Using a series of modified amino acids, we also found similarly high correlations (R2 > 0.9600) between Inductive Indexes and two wholly independent chemical properties: i) the pKa values of ionizable side-chains and, ii) the fluorescence response of the indole group of tryptophan. After assessing the applicability of the method of calculation at the amino acid level, we extended our study to tryptophan-containing peptides and established that inductive contributions of neighboring side-chains are transmitted through peptide bonds. We discuss possible contributions to the study of proteins.

Paraspecific neutralization of the venom of African species of cobra by an equine antiserum against Naja melanoleuca: a comparative study.

Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the venoms and the paraspecific median effective doses (ED50) of the antiserum calculated using three methods: Spearman-Kärber and Probit (currently recommended by the World Health Organization), and non-linear regression. An ample--but not complete--spectrum of paraspecific neutralization of lethality was observed against both spitting and non-spitting species of African Naja with a clearly more efficient neutralization of the more potent venoms, the implications of which are discussed. The median lethal and effective doses calculated by the three methods are remarkably consistent and may warrant consideration of non-linear regression methods for the calculation of venom lethality and antivenom potency by venom/antivenom researchers and producers.

Coupled Response of Membrane Hydration with Oscillating Metabolism in Live Cells: An Alternative Way to Modulate Structural Aspects of Biological Membranes?

We propose that active metabolic processes may regulate structural changes in biological membranes via the physical state of cell water. This proposition is based on recent results obtained from our group in yeast cells displaying glycolytic oscillations, where we demonstrated that there is a tight coupling between the oscillatory behavior of glycolytic metabolites (ATP, NADH) and the extent of the dipolar relaxation of intracellular water, which oscillates synchronously. The mechanism we suggest involves the active participation of a polarized intracellular water network whose degree of polarization is dynamically modulated by temporal ATP fluctuations caused by metabolism with intervention of a functional cytoskeleton, as conceived in the long overlooked association-induction hypothesis (AIH) of Gilbert Ling. Our results show that the polarized state of intracellular water can be propagated from the cytosol to regions containing membranes. Since changes in the extent of the polarization of water impinge on its chemical activity, we hypothesize that metabolism dynamically controls the local structure of cellular membranes via lyotropic effects. This hypothesis offers an alternative way to interpret membrane related phenomena (e.g., changes in local curvature pertinent to endo/exocytosis or dynamical changes in membranous organelle structure, among others) by integrating relevant but mostly overlooked physicochemical characteristics of the cellular milieu.

Characterization of a new polyvalent antivenom (Antivipmyn Africa) against African vipers and elapids.

As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn((R)) Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab')(2)-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.

Study of the efficacy of the black stone on envenomation by snake bite in the murine model.

The black stone (BS) has been used since antiquity to treat envenomations. Since no actual clinical trial has ever been performed we used an experimental approach to evaluate its efficacy against the venoms of Bitis arietans, Echis ocellatus and Naja nigricollis. Local application of BS after intramuscular venom injection had no demonstrable effect on the outcome of envenomationa and it did not change the LD(50) of B. arietans venom. Our results show that, contrary to widespread belief, no efficacy to treat envenomation may be expected of the BS.

The dynamics of intracellular water constrains glycolytic oscillations in Saccharomyces cerevisiae.

We explored the dynamic coupling of intracellular water with metabolism in yeast cells. Using the polarity-sensitive probe 6-acetyl-2-dimethylaminonaphthalene (ACDAN), we show that glycolytic oscillations in the yeast S. cerevisiae BY4743 wild-type strain are coupled to the generalized polarization (GP) function of ACDAN, which measures the physical state of intracellular water. We analysed the oscillatory dynamics in wild type and 24 mutant strains with mutations in many different enzymes and proteins. Using fluorescence spectroscopy, we measured the amplitude and frequency of the metabolic oscillations and ACDAN GP in the resting state of all 25 strains. The results showed that there is a lower and an upper threshold of ACDAN GP, beyond which oscillations do not occur. This critical GP range is also phenomenologically linked to the occurrence of oscillations when cells are grown at different temperatures. Furthermore, the link between glycolytic oscillations and the ACDAN GP value also holds when ATP synthesis or the integrity of the cell cytoskeleton is perturbed. Our results represent the first demonstration that the dynamic behaviour of a metabolic process can be regulated by a cell-wide physical property: the dynamic state of intracellular water, which represents an emergent property.

North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

Report of the 2nd International Conference on Envenomations in Africa (Deuxième Colloque International sur les Envenomations en Afrique).

An international meeting on envenomations in Africa was held in Cotonou, Republic of Benin, the 19th and 20th of September, 2004, to gather clinical practitioners, epidemiologists, health authorities and antivenom producers to present and discuss the most recent available data on the prevalence of snake envenomations in Africa. The meeting concluded with a round table which had the objective of evaluating the problems facing the epidemiology and therapy of envenomations by African snakes and making some general recommendations at the level of national health authorities, researchers, medical practitioners, and international antivenom producers.

Tight coupling of metabolic oscillations and intracellular water dynamics in Saccharomyces cerevisiae.

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D2O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process.

Genetic and enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback spiders Loxosceles boneti and Loxosceles reclusa.

In this study we report the isolation and characterization of several sphingomyelinase D isoforms from the venoms of the North American spiders Loxosceles boneti and Loxosceles reclusa, from Mexico and the United States, respectively. We have measured their enzymatic activity, their capacity to induce necrotic lesions in rabbits, cloned the cDNAs coding for the mature forms of two of the isoforms from L. boneti and two of L. reclusa based on N-terminal sequence information of the purified proteins, and performed a comprehensive comparison of the sequence data generated by us with that reported for other sphingomyelinase genes to date.

Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa.

BACKGROUND: In Guinea Elapids are responsible for 20% of envenomations. The associated case fatality rate (CFR) ranged 15-27%, irrespective of treatment. RESULTS: We studied 77 neurotoxic envenomations divided in 3 groups: a set of patients that received only traditional or symptomatic treatments, and two other groups that received either 2 or 4 initial vials of Antivipmyn® Africa renewed as necessary. CFR was 27.3%, 15.4% and 17.6%, respectively. Although antivenom treatment was likely to reduce CFR, it didn't seem to have an obvious clinical benefit for the patients, suggesting a low treatment efficacy. Mean delay to treatment or clinical stages were not significantly different between the patients who recovered and the patients who died, or between groups. Interpretation of these results is complicated by the lack of systematic studies under comparable conditions. Of particular importance is the absence of assisted ventilation, available to patients in all the other clinical studies of neurotoxic envenomation. CONCLUSION: The apparent lack of clinical benefit may have several causes. The hypothesis of a limited therapeutic window, i.e. an insufficient formation of antigen-antibody complexes once toxins are bound to their targets and/or distributed beyond the reach of antivenom, should be explored.

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Report of the 4th International Conference on Envenomations by Snakebites and Scorpion Stings in Africa, Dakar, April 25-29, 2011.

The authors present a summary of the proceedings and the recommendations of the the 4th International Conference on Envenomations by Snakebites and Scorpion Stings in Africa, held from April 25th to 29th in Dakar. After a two day training workshop for Senegalese health personnel on the most relevant aspects of the management of envenomations, about 270 participants met to share their experiences in the field. Nearly a hundred oral and poster contributions concerning the epidemiology of snakebites and scorpion stings in Africa, the composition and action of venoms, as well as the manufacture and use of antivenoms, were presented and discussed. The last day was devoted to an institutional debate joining experts, representatives of national health authorities and concerned professionals (physicians, pharmacists, nurses and traditional healers) as well as members of the pharmaceutical industry, to discuss and elaborate a set of recommendations. It was agreed that it is necessary to improve knowledge of the epidemiological situation by case reporting. Quality control of anivenoms and procedures for their registration at the level of national health authorities should aim at improving the distribution of safe and effective antivenoms in peripheral health centers, which bear the heaviest burden of cases. It was also recommended that adequate training of health personnel in all aspects of medical management of envenomations should constitute a priority. Finally, financing mechanisms to ensure an equitable distribution of resources must be sought, as well as the constitution of a network of African experts were discussed at length.