Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.     

Outside the Unusual Cell Wall of the Hyperthermophilic Archaeon Aeropyrum pernix K1

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.The archaea are a unique group of organisms that share properties with both the eukarya and bacteria. For a long time, archaeal life was considered to be limited to extreme environments such as high temperature, alkaline and acidic hot springs, anaerobic sediments, and highly saline environments. In the last decade, by the use of the archaeal 16 S rRNA gene as a molecular marker in microbial surveys (1), numerous mesophilic species have also been detected (2). Archaea have been found frequently and sometimes closely associated with bacterial and eukaryotic host cells, including humans. One of the most intriguing aspects of archaea is their unusual barrier between the inner cell material and the cellular environment, i.e. their cell membrane. Biosynthesis of archaeal cell wall has been a subject of interest for a long time. Most of the archaeal species characterized so far have a single chemically distinct cell membrane, which differs considerably from their eukaryotic and bacterial counterparts (3). The ether-type polar lipid surface is covered by a surface layer (S-layer)¹ composed of glycoproteins crystallized in regular two-dimensional lattices with hexagonal or tetragonal symmetry (4, 5). The structural characterization of the S-layer (6, 7) and S-layer-embedded archaeal cellular appendices such as flagella (8), pili, and hami (7, 9) associated with a diverse arsenal of cellular functions like motility, cell-cell communication, signaling, adherence, and nutrient uptake, has been the subject of an increasingly significant number of studies. Protein secretion mechanisms through this unusual cell membrane have been mainly addressed by way of comparative genomics studies (10–12) and by genomic identification and characterization of signal peptidases (13, 14). Archaeal extracellular and cell membrane proteins have been predicted because of the presence of a tripartite N-terminal signal motif essential for protein secretion and subsequently cleaved by signal peptidases from the protein (11, 14, 15). In archaea three different signal peptidases have been identified and characterized so far (13): signal peptidase I is responsible for the cleavage of secretory signal peptides from the majority of secreted proteins, class III signal peptidase is responsible for processing signal peptides from preflagellins and some sugar-binding proteins (11), and signal peptide peptidase is responsible for the hydrolysis of signal peptides following protein secretion. No signal peptidase II homolog in archaea has been described to date. Four distinct pathways have been proposed for archaeal protein export: the main “Sec” system, the twin arginine translocation or “Tat” pathway (12), the ATP-binding cassette (ABC) transport system (16), and the type IV prepilin-like pathway (11). Moreover, proteins without signal peptides could also be secreted by using nonspecific and/or currently unknown mechanisms. Despite the similarities in protein translocation mechanisms between the three domains of life, genome analyses also shed light on unique archaeal characteristics, suggesting that our current knowledge regarding secreted proteins and secretion mechanisms in archaea remains limited (10). It is apparent that the lack of experimental data at the proteome level has become the bottleneck for the further understanding of the existence of novel secretion mechanisms in archaea (15).To date, the genome sequences of eight hyperthermophiles, including the crenarchaeon Aeropyrum pernix K1, have been determined. A. pernix K1, isolated from a coastal solfataric thermal vent on the Kodakara-Jima Island in Japan (17), is the first reported obligate aerobic and neutrophilic hyperthermophilic archaeon with an optimal growth temperature between 90 and 95 °C. The spherical shaped cells of A. pernix are ∼1 μm in diameter, lack a rigid cell wall, and are covered by an S-layer with hexagonal symmetry. A. pernix, like other extreme thermophiles and acidophiles, possesses a particularly thick cell membrane that acts as a protective barrier, conferring to it the ability to function in the extreme environment in which it thrives. The lipids of A. pernix are different from those of anaerobic sulfur-dependent hyperthermophiles; they lack tetraether lipids and the direct linkage of inositol and sugar moieties (18). A. pernix K1 contains a 1.6-Mbp chromosome that has been sequenced; it comprises 1700 annotated genes. By using different proteomics approaches, the proteome of A. pernix K1 has recently been analyzed, leading to the identification of 704 proteins (41% of total ORFs) (19). In this work we performed proteomics analysis of the cell surface and extracellular protein fractions purified from A. pernix K1 to define proteins targeted to the cell secretome. We also analyzed the complete predicted proteome of A. pernix K1 by in silico signal peptide and cellular localization prediction tools and compared the experimentally obtained data set with the predicted secretome.     

Morpho-functional characterization of cultured pigment cells fromRana esculenta L. Liver

Summary A simple method to isolate and culture liver pigment cells fromRana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells fromRana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance. This research was partly supported by grant of Ministero della Pubblica Istruzione, Ricerca Scientifica.     

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.     

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.     

<Emphasis Type="Italic">Scenedesmus vacuolatus</Emphasis> cultures for possible combined laccase-like phenoloxidase activity and biodiesel production

A key aspect of the industrial development of microalgal production processes is the excessive cost of biomass production. A solution is a combination of biodiesel production and wastewater treatment. The microalga Scenedesmus has a high lipid content and a potential extracellular phenoloxidase activity, which could improve the phycoremediation of phenolic pollutants. In this work, the most suitable growth conditions to obtain this twofold aim were analyzed. First, different strains of Scenedesmus vacuolatus microalga were tested at different pH, salinity and CO₂ concentration in the gas phase. The two most promising strains were then cultivated in autotrophic and heterotrophic conditions, and were investigated in terms of efficient nitrogen removal, fatty acid profile and maximized extracellular phenoloxidase activity in the medium. The results showed two extreme conditions: (1) biomass productivity doubled when photobioreactors were sparged with 5% CO₂ supplemented air with respect to cultures sparged with air (the steady state values of strain 53 were 0.138 g L^?1 day^?1 in the presence of air, and 0.243 in the presence of CO₂ addition), and N-starvation under 5% CO₂enhanced the transesterified fraction of lipids (strain 53 FAME fraction in the presence of N-starvation was 33%, in the presence of nitrogen FAME fraction was 22%); (2) phenoloxidase activity was completely suppressed by presence of 5% CO₂ in the gas phase (strain 53 0.21 U mL^?1), indicating clear catabolite repression for the induction of this enzyme in the algal metabolism.