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.

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 calculation of the magnitude of inductive effects for non-aromatic molecules. The value obtained by the algorithm is called the and we observed a high correlation (R = 0.

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.

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.

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.

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.

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.

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.

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.

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.

Myotoxicity and nephrotoxicity by Micrurus venoms in experimental envenomation.

Micrurus venoms are essentially neurotoxic but other activities, such as myotoxicity, may be apparent under experimental conditions. Although this myotoxicity has been occasionally reported, there are no studies addressing it systematically across the genus, particularly in its relationship to other systemic manifestations such as renal impairment. The lethal potency of Micrurus fulvius, Micrurus nigrocinctus, Micrurus surinamensis, Micrurus altirostris, Micrurus balyocoriphus and Micrurus pyrrhocryptus venoms determined by us were in the range described for the genus and all venoms exhibited phospholipase activity, albeit at significantly different levels.

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.

Neutralization of Bothrops alternatus regional venom pools and individual venoms by antivenom: a systematic comparison.

In this study we report that variation in lethality, hemorrhagic potency and procoagulation between individual samples of Bothrops alternatus venom from a single region, and variation between regional pools at the national level are comparable in range. Furthermore, the range of relative neutralization potencies of individual venoms within a region overlaps, and sometimes exceeds, the range of neutralization of regional venom pools throughout the country. Thus, the potency of neutralization of a national venom pool is poorly predictive of the potencies of neutralization of its constituent regional venom pools and, furthermore, the potency of neutralization of a regional venom pool is poorly predictive of the potencies of neutralization of its individual venom constituents.

Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: a study of paraspecificity.

We conducted an extensive study of neutralization of lethality of 11 species and one subspecies of snakes of the genus Vipera, and of five species of Macrovipera, by two experimental equine antisera. One antiserum was a trivalent preparation raised against the venoms of Vipera aspis aspis, Vipera berus berus and Vipera ammodytes ammodytes; the other was a pentavalent preparation that also included venoms of Vipera (now Montivipera) xanthina and Macrovipera lebetina obtusa. We measured specific neutralization of lethality against all venoms included in the immunization schemes, and paraspecific neutralization against the venoms of Vipera ammodytes montandoni, Vipera (Montivipera) bornmuelleri, Vipera latastei, Vipera (Mo.

Methodology of clinical studies dealing with the treatment of envenomation.

A total of 142 clinical studies have been devoted to the treatment of envenomations, of which 115 address snake bites, 20 scorpion stings, and 8 other animals (one addresses both snake and spider envenomation). Antivenom use was studied in 118, of which 82 addressed efficacy, 43 evaluated safety, 23 studied dosage and 8 explored other issues. Besides anecdotal clinical reports, three classes of clinical studies are distinguished: (a) observational clinical studies (55 of the total) which analyze series of cases, (b) comparative clinical studies (36) which compare therapeutic products or treatment regimens without a gold standard for comparison and (c) randomized clinical trials (RCT, 51).

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.

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.

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.

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.

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.

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.

Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids.

The Sec61alpha protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61alpha subunit (EhSec61alpha), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids and its effects on cell proliferation, subcellular distribution of two virulence factors, and the ability of the trophozoites to cause liver abscess in hamsters. Although Sec61alpha levels are specifically decreased in antisense PNA-treated trophozoites, which proliferate more slowly than the controls, mobilization of the cysteine protease 5 and amoebapore to the cell surface is not significantly impeded and the capacity to induce liver abscess in hamsters is largely unaffected.

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.

Entamoeba histolytica: intracellular distribution of the proteasome.

We have studied the intracellular distribution of proteasome subunits, corresponding to the catalytic (20S) core and the regulatory (19S) cap, in the extracellular protozoan parasite Entamoeba histolytica. Contrary to all cell types described to date, notably mammalian and yeast, in which the proteasome is found in the nucleus and actively imported into it, microscopic analysis and subcellular fractionation of E. histolytica trophozoites show that the proteasome is absent from the nucleus of these cells.