A model for the origin of stable protocells in a primitive alkaline ocean

When a mixture of the eighteen proteinous amino acids are suitably heated in the dry state with seawater salts, a copolyamino acid results. One fraction of this polymer is found, through isoelectric focusing, to consist of a mixture of acidic and basic proteinoids, each of sharply limited heterogeneity. When one fraction of the seawater proteinoid is dissolved in hot water, and the solution is cooled, proteinoid microspheres result. These have properties in common with simpler types, but are also stable at pH values to 9, in common with microspheres prepared by mixing acidic and basic proteinoids. These processes thus constitute a simple model for the origin of a protocell stable in a primitive alkaline ocean.     

Interactions between diverse froteinoids and microspheres in simulation of primordial evolution

Experiments demonstrating an incorporation of different enzymelike activities into a single preparation of proteinoid microspherss provide a conceptual basis for the primitive lengthening of protometabolic pathways. An enhancement of one enzymelike activity by another proteinoid in the same microsphere has been found. This effect, plus the pathway-lengthening propensity of combinations of microspheres, indicates selective advantages contributing to adaptive protoselection.Data reported in this paper also bring into purview the concept of internally controlled variation. Inferences are derived for the origin of protosexuality in protocells.When allowance is made for a closer relationship to the environment than that needed in contemporary selection, the fundamental mechanistic requirements of protoevolution are regarded as met by the proteinoid microsphere.     

Formation of proteinoid microspheres under simulated prebiotic atmospheres and individual gases.

The formation of microspheres from acidic and basic proteinoids was attempted under simulated prebiotic atmospheres and constituent gases thereof. Both types of proteinoid yielded microspheres under carbon dioxide, carbon monoxide, methane, hydrogen sulfide, hydrogen, nitrogen, and oxygen (tested separately) and also under nitrogen-carbon dioxide atmospheres; higher proportions of carbon dioxide resulted in fewer spheres from basic proteinoid. Neither type of proteinoid formed spheres on 10-minute exposure to ammonia or methane-hydrogen-ammonia atmospheres. (Brief exposure resulted in spheres from basic proteinoid.) The effects, both qualitative and quantitative, were indicated by control experiments to be due to pH, rather than to the specific gas (or ion). The results suggest that the proteinoid microsphere model for protocells is applicable under a variety of possible prebiotic atmospheres, with some restrictions imposed by pH.     

Proteinoid microspheres more stable in hot than in cold water

The occurrence of organisms of primitive appearance near submarine hydrothermal vents has indicated sea-floor conditions that are like those under which proteinoid microspheres are produced in the laboratory. Experimental examination of the question of whether some proteinoid microspheres might be stable in hot water has revealed proteinoids that are soluble in cold water but precipitate on heating. Unanswered questions are discussed.     

From proteinoid microsphere to contemporary cell: Formation of internucleotide and peptide bonds by proteinoid particles

Proteinoid microspheres of appropriate sorts promote the conversion of ATP to adenine dinucleotide and adenine trinucleotide. Other microparticles composed of basic proteinoid and enzymically synthesized poly A cause the conversion of ATP and phenylalanine to various peptides of phenylalanine. When viewed in a context with the origin and properties of proteinoid microspheres, these results model the origin from a protocell of a more contemporary type of cell able to synthesize its own polyamino acids and polynucleotides. Related earlier experiments explain in part the origin of the genetic code and mechanism.     

Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

When lysine-rich proteinoid, which catalyzes the formation of peptides from amino acids and ATP, is complexed with acidic proteinoid to form microspheres of mixed constitution, the normal synthesis by basic proteinoid alone is multiplied several-fold. The product consists not only of small peptides but also of a high-molecular-weight fraction of substituted proteinoid.Suspensions of particles of lysine-rich proteinoid complexed with polyadenylic acid catalyze the synthesis of peptides from each of the amino acids tested with ATP. When equimolar solutions of mixtures of glycine and phenylalanine with ATP are tested in suspensions of complexes of lysine-rich proteinoid and each of various polyribonucleotides, both homopeptides and heteropeptides are produced. Glycylphenylalanine or phenylalanylglycine is the principal product; the preference is related to which polyribonucleotide is in the complex.The rate of conversion of amino acid to peptide is a function of whether ATP is added in a single batch or in repeated amounts adding to the same amount as in the single batch. Related experiments indicate a relatively rapid initial rate of decay of ATP in this system. These results are discussed relative to the mechanisms for continuous generation in modern organisms, as are the results in peptide formation.     

Electrical excitability of proteinoid microspheres composed of basic and acidic proteinoids

Material flow equilibration, an endogenous interaction adjustment process underlying fulfillment of material flow continuity during material self-assembly, also underlies electrical excitability observed in the proteinoid microspheres as simulated models of the protocell. The spikings of the membrane potentials are attributed to a singular character of the interaction rate coefficients measuring the strengths of the coupling between basic and acidic proteinoids, in which the rates change singularly with time due to material flow equilibration.     

Coacervate-like microspheres from lysine-rich proteinoid

Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate dtoplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.     

Proteinoid microspheres and the process of prebiological photophosphorylation

A chemical model of prebiological photophosphorylation with participation of hemoproteinoid microspheres, mixed microspheres containing bonded riboflavin and microspheres obtained from glycine rich proteinoids was studied. The illumination of aqueous solutions containing microspheres, K₂HPO₄, ADP and electron acceptor leads to an increase of ATP concentration and to a decrease of concentration of inorganic phosphate. Initial photochemical reactions with participations of proteinoid microspheres could have evolved in the course of chemical evolution and led to the emergence of the photophosphorylation in its modern biochemical form.     

Assembly of microspheres from acidic proteinoids and histones or histone-like proteinoids

Basic polyamino acids, whether organismic or synthetic in origin, alter the morphology of proteinoid microspheres in which they are included. The particles obtained with histone are essentially indistirguishable under the light microscope from those obtained with proteinoids of histone-like composition (histonoids). In each instance, structures of morphological complexity similar to those of some microfossils are obtained.     

Activation of glycine by ATP,a divalent cation,and proteinoid microspheres

The activation of glycine to yield glycyl hydroxamate has been studied in the absence of enzymes. Activation with ATP in aqueous solution requires only a divalent metal cation. ATP is far more active than other nucleoside triphosphates; AMP and pyrophosphate are inactive. The pH optimum is 4 to 5; activation at pH 7 is most enhanced in the presence of proteinoid microspheres.     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

Characterization of patocytosis: endocytosis into macrophage surface-connected compartments

Previously, we described a unique macrophage endocytosis pathway in which aggregated low density lipoproteins and microcrystalline cholesterol induce and enter a labyrinth of membrane-bound compartments that remain connected to the cell surface. We now show that certain types of non-lipid particles such as polystyrene microspheres and colloidal gold also induce and enter macrophage surface-connected compartments (SCC), a process we call patocytosis. A common property among particles that stimulate patocytosis is their hydrophobic nature. Both aggregated LDL and microcrystalline cholesterol that we showed previously to stimulate patocytosis are hydrophobic. We now show that hydrophobic polystyrene microspheres and gold particles but not their hydrophilic counterparts triggered patocytosis. Uptake by patocytosis was limited to hydrophobic polystyrene microsphere particles less than 0.5 micron in diameter. Hydrophobic polystyrene microspheres greater than this size entered macrophages by phagocytosis. Actin-independent capping of hydrophobic polystyrene microspheres on the plasma membrane preceded actin-dependent uptake of the microspheres into SCC. Sequential rounds of microsphere uptake into SCC over two successive days could occur. There was some mixing of initial and subsequently accumulated microspheres in SCC. SCC formed from plasma membrane invaginations that connected with spaces created by unfolding of stacks of internal microvilli. Microsphere transport from plasma membrane invaginations into these spaces was inhibited by primaquine. Patocytosis is a unique endocytic process in macrophages triggered by small hydrophobic particles that provides a mechanism to sequester large amounts of these materials within a labyrinth of SCC.     

Synthesis of life in the lab? Defining a protoliving system

The synthesis of a living system in the lab has been judged by a number of critics as partly attained by the proteinoid microsphere because of its primitive properties of metabolism, growth, and reproduction. These same critics, however, judge the organism as not alive, or as being 50 to 75 percent alive (Baltscheffsky and Jurka, 1984), owing to the absence of a nucleic acid genetic coding mechanism. The experiments in retracing evolution suggest, however, that the self-sequencing of amino acids was the evolutionary precursor of modern nucleic acid templating; the genetic memory is the molecule. The proteinoid microsphere is not a modern living system, but does represent at least a protoliving system (Fox and Dose, 1972). Berra (1990, p. 75) has commented on other difficulties in defining a protoliving system. In Berra's opinion, metabolism, reproduction, responsiveness to stimuli, and cellularity constitute or describe aliveness. These properties characterize proteinoid microspheres. A number of experiments demonstrate that amino acids in aminoacyl adenylates yield specific products, whereas nucleotides are without effect. For this and related reasons, especially the demonstrated self-sequencing of amino acids when they are warmed, resultant bio-functional properties of self-assembled microstructures, and demonstrated self-sequencing of amino acids in modern systems, the results appear to bridge from the chemical era to the biological period. All the above emerges from a departure in style of research (Young, 1984; Pauling and Zuckerkandl, 1972). The latter authors said, "It appears likely that biogenesis is the passage from a 'non-living system' existing in a large number of states to a 'living' system also existing in a large number of states."(ABSTRACT TRUNCATED AT 250 WORDS)     

Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides.

Major variables in interactions between basic thermal proteinoids and homopolyribonucleotides were magnesium concentration in solution (0–40 mM) and mol% lysine in the proteinoid (16–55%). The formation of microparticles was monitored both by the turbidity and by the mass of precipitate formed. Under some conditions, only, was the turbidity reading a reliable indication of the amount of precipitate. Increasing concentration of Mg²⁺ tended to displace proteinoid from the complex with polynucleotide. Of 4 polynucleotides, only polyguanylic acid showed an enhanced precipitation of proteinoid in the presence of Mg²⁺, and then only with those having high lysine contents. At high lysine contents, the amount of proteinoid in the precipitate was inversely proportional to the lysine content of the proteinoids, probably due to decreased sidechain interactions. The precipitation with polynucleotides is partly a function of the amino acid composition of the proteinoid; therefore the interaction of thermal proteinoids with polynucleotides appears to be a tool that can be used to study specificities of interactions between proteins and nucleic acids.     

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-b-poly(D,L-lactide) microspheres for protein delivery

Temperature-sensitive diblock copolymers, poly(N-isopropylacrylamide)-b-poly(D,L-lactide) (PNIPAAm-b-PLA) with different PNIPAAm contents were synthesized and utilized to fabricate microspheres containing bovine serum albumin (BSA, as a model protein) by a water-in-oil-in-water double emulsion solvent evaporation process. XPS analysis showed that PNIPAAm was a dominant component of the microspheres surface. BSA was well entrapped within the microspheres, and more than 90% encapsulation efficiency was achieved. The in vitro degradation behavior of microspheres was investigated using SEM, NMR, FTIR, and GPC. It was found that the microspheres were erodible, and polymer degradation occurred in the PLA block. Degradation of PLA was completed after 5 months incubation in PBS (pH 7.4) at 37 degrees C. A PVA concentration of 0.2% (w/v) in the internal aqueous phase yielded the microspheres with an interconnected porous structure, resulting in fast matrix erosion and sustained BSA release. However, 0.05% PVA produced the microspheres with a multivesicular internal structure wrapped with a dense skin layer, resulting in lower erosion rate and a biphasic release pattern of BSA that was characterized with an initial burst followed by a nonrelease phase. The microspheres made from PNIPAAm-b-PLA with a higher portion of PNIPAAm provided faster BSA release. In addition, BSA release from the microspheres responded to the external temperature changes. BSA release was slower at 37 degrees C (above the LCST) than at a temperature below the LCST. The microspheres fabricated with PNIPAAm-b-PLA having a 1:5 molar ratio of PNIPAAm to PLA and 0.2% (w/v) PVA in the internal aqueous phase provided a sustained release of BSA over 3 weeks in PBS (pH 7.4) at 37 degrees C.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

The relationship of K+ efflux at the inner surface of the isolated frog skin epithelium to the short circuit current.

Isolated frog skins (without chorion) were incubated with 42K+ Ringers' solution, bathing the internal surface for 2 h. All the K+ contained in the frog skin was equilibrated in specific activity with external 42K+. The kinetics of the washout of 42K+ from the internal surface of the skin exhibits one fast and one slow exponential component. Amiloride reduces the release of 42K+ corresponding to both components without affecting the K+ content of the skin. Ouabain increases the loss of 42K+ of the slow component by 200%. Since the total K+ in the skin decreases to 25% of its original value both compartments are affected. The results suggest that two distinct functional compartments exist defined by two 42K+ release ratios and that because of the large K+ contents of these compartments both are intracellular. The relation with the transepithelial Na+ transport and the morphological identification of these compartments is discussed.     

Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge⁻¹ day⁻¹. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769–773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH₄-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea. However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.