Open Questions on the Origin of Life at Anoxic Geothermal Fields

We have recently reconstructed the ??hatcheries?? of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells (Mulkidjanian et al. Proc Natl Acad Sci U S A 109:E821?C830, 2012). These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K⁺, Zn²⁺, Mn²⁺, and phosphate. Thus, protocells must have evolved in habitats with a high K⁺/Na⁺ ratio and relatively high concentrations of Zn, Mn and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under an anoxic, CO₂-dominated atmosphere, the ionic composition of pools of cool, condensed vapor at anoxic geothermal fields would resemble the internal milieu of modern cells. Such pools would be lined with porous silicate minerals mixed with metal sulfides and enriched in K⁺ ions and phosphorous compounds. Here we address some questions that have appeared in print after the publication of our anoxic geothermal field scenario. We argue that anoxic geothermal fields, which were identified as likely cradles of life by using a top-down approach and phylogenomics analysis, could provide geochemical conditions similar to those which were suggested as most conducive for the emergence of life by the chemists who pursuit the complementary bottom-up strategy.     

Strain tunes proteolytic degradation and diffusive transport in fibrin networks

Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials.     

Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail

The mechanism of the Golgi-to-ER transport of Golgi glycosyltransferases is not clear. We utilize a cell line expressing the core 2 N-acetylglucosaminyltransferase-M (C2GnT-M) tagged with c-Myc to explore this mechanism. By immunoprecipitation using anti-c-Myc antibodies coupled with proteomics analysis, we have identified several proteins including non-muscle myosin IIA (NMIIA), heat shock protein (HSP)-70 and ubiquitin activating enzyme E1 in the immunoprecipitate. Employing yeast-two-hybrid analysis and pulldown experiments, we show that the C-terminal region of the NMIIA heavy chain binds to the 1-6 amino acids in the cytoplasmic tail of C2GnT-M. We have found that NMIIA co-localizes with C2GnT-M at the periphery of the Golgi. In addition, inhibition or knockdown of NMIIA prevents the brefeldin A-induced collapse of the Golgi as shown by the inhibition of the migration of both Giantin, a Golgi matrix protein, and C2GnT-M, a Golgi non-matrix protein, to the ER. In contrast, knockdown of HSP70 retains Giantin in the Golgi but moves C2GnT-M to the ER, a process also blocked by inhibition or knockdown of NMIIA. Also, the intracellular distribution of C2GnT-M is not affected by knockdown of β-coatomer protein with or without inhibition of HSPs, suggesting that the Golgi-to-ER trafficking of C2GnT-M does not depend on coat protein complex-I. Further, inhibition of proteasome results in accumulation of ubiquitinated C2GnT-M, suggesting its degradation by proteasome. Therefore, NMIIA and not coat protein complex-I is responsible for transporting the Golgi glycosyltransferase to the ER for proteasomal degradation. The data suggest that NMIIA is involved in the Golgi remodeling.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase

Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Proton transfer in the photosynthetic reaction center of Blastochloris viridis

Photosynthetic reaction centers of Blastochloris viridis require two quanta of light to catalyse a two-step reduction of their secondary ubiquinone Q(B) to ubiquinol. We employed capacitive potentiometry to follow the voltage changes that were caused by the accompanying transmembrane proton displacements. At pH 7.5 and 20 degrees C, the Q(B)-related voltage generation after the first flash was contributed by a fast, temperature-independent component with a time constant of approximately 30 micros and a slower component of approximately 200 micros with activation energy (E(a)) of 50 kJ/mol. The kinetics after the second flash featured temperature-independent components of 5 micros and 200 micros followed by a component of 600 micros with E(a) approximately 60 kJ/mol.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

Local neurons play key roles in the mammalian olfactory bulb

Over the past few decades, research exploring how the brain perceives, discriminates, and recognizes odorant molecules has received a growing interest. Today, olfaction is no longer considered a matter of poetry. Chemical senses entered the biological era when an increasing number of scientists started to elucidate the early stages of the olfactory pathway. A combination of genetic, biochemical, cellular, electrophysiological and behavioral methods has provided a picture of how odor information is processed in the olfactory system as it moves from the periphery to higher areas of the brain. Our group is exploring the physiology of the main olfactory bulb, the first processing relay in the mammalian brain. From different electrophysiological approaches, we are attempting to understand the cellular rules that contribute to the synaptic transmission and plasticity at this central relay. How olfactory sensory inputs, originating from the olfactory epithelium located in the nasal cavity, are encoded in the main olfactory bulb remains a crucial question for understanding odor processing. More importantly, the persistence of a high level of neurogenesis continuously supplying the adult olfactory bulb with newborn local neurons provides an attractive model to investigate how basic olfactory functions are maintained when a large proportion of local neurons are continuously renewed. For this purpose, we summarize the current ideas concerning the molecular mechanisms and organizational strategies used by the olfactory system to encode and process information in the main olfactory bulb. We discuss the degree of sensitivity of the bulbar neuronal network activity to the persistence of this high level of neurogenesis that is modulated by sensory experience. Finally, it is worth mentioning that analyzing the molecular mechanisms and organizational strategies used by the olfactory system to transduce, encode, and process odorant information in the olfactory bulb should aid in understanding the general neural mechanisms involved in both sensory perception and memory. Due to space constraints, this review focuses exclusively on the olfactory systems of vertebrates and primarily those of mammals.     

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.