Angeles Sánchez-Pérez

Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na+ absorption; hence its function is essential for maintaining Na+ and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1K179M, decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser342 and Ser428, completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na+ absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria.

Antimicrobial peptides (AMPs) are short peptidic molecules produced by most living creatures. They help unicellular organisms to successfully compete for nutrients with other organisms sharing their biological niche, while AMPs form part of the immune system of multicellular creatures. Thus, these molecules represent biological weapons that have evolved over millions of years as a result of an escalating arms race for survival among living organisms. All AMPs share common features, such as a small size, with cationic and hydrophobic sequences within a linear or cyclic structure. AMPs can inhibit or kill bacteria at micromolar concentrations, often by non-specific mechanisms; hence the appearance of resistance to these antimicrobials is rare. Moreover, AMPs can kill antibiotic-resistant bacteria, including insidious microbes such as Acinetobacter baumannii and the methicillin-resistant Staphylococcus aureus. This review gives a detailed insight into a selection of the most prominent and interesting AMPs with antibacterial activity. In the near future AMPs, due to their properties and despite their ancient origin, should represent a novel alternative to antibiotics in the struggle to control pathogenic microorganisms and maintain the current human life expectancy.

Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b5 as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b5, provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the “IDO family”. Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-d-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

Construction of a novel Pichia pastoris strain for production of xanthophylls.

In this study, we used the yeast carotenogenic producer Pichia pastoris Pp-EBIL strain, which has been metabolically engineered, by heterologously expressing β-carotene-pathway enzymes to produce β-carotene, as a vessel for recombinant astaxanthin expression. For this purpose, we designed new P. pastoris recombinant-strains harboring astaxanthin-encoding genes from carotenogenic microorganism, and thus capable of producing xanthophyllic compounds. We designed and constructed a plasmid (pGAPZA-WZ) containing both the β-carotene ketolase (crtW) and β-carotene hydroxylase (crtZ) genes from Agrobacterium aurantiacum, under the control of the GAP promoter and containing an AOX-1 terminator. The plasmid was then integrated into the P. pastoris Pp-EBIL strain genomic DNA, producing clone Pp-EBILWZ. The recombinant P. pastoris (Pp-EBILWZ) cells exhibited a strong reddish carotenoid coloration and were confirmed, by HPLC, to produce not only the previous described carotenoids lycopene and β-carotene, but also de novo synthesized astaxanthin.

Recent patents on microbial proteases for the dairy industry.

This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.

Cell aggregations in yeasts and their applications

Yeasts can display four types of cellular aggregation: sexual, flocculation, biofilm formation, and filamentous growth. These cell aggregations arise, in some yeast strains, as a response to environmental or physiological changes. Sexual aggregation is part of the yeast mating process, representing the first step of meiotic recombination. The flocculation phenomenon is a calcium-dependent asexual reversible cellular aggregation that allows the yeast to withstand adverse conditions. Biofilm formation consists of multicellular aggregates that adhere to solid surfaces and are embedded in a protein matrix; this gives the yeast strain either the ability to colonize new environments or to survive harsh environmental conditions. Finally, the filamentous growth is the ability of some yeast strains to grow in filament forms. Filamentous growth can be attained by two different means, with the formation of either hyphae or pseudohyphae. Both hyphae and pseudohyphae arise when the yeast strain is under nutrient starvation conditions and they represent a means for the microbial strain to spread over a wide area to survey for food sources, without increasing its biomass. Additionally, this filamentous growth is also responsible for the invasive growth of some yeast.

Cloning and Characterization of the Beer Foaming Gene CFG1from Saccharomyces pastorianus

Foam production is an essential characteristic of beer, generated mainly from the proteins present in the malt and, to a minor extent, from the mannoproteins in brewers yeast cell walls. Here, we describe the isolation and characterization of the novel fermentation gene CFG1 (Carlsbergensis foaming gene) from Saccharomyces pastorianus. CFG1 encodes the cell wall protein Cfg1p, a 105 kDa protein highly homologous to Saccharomyces cerevisiae cell wall mannoproteins, particularly those involved in foam formation, such as Awa1p and Fpg1p. Further characterization of Cfg1p revealed that this novel protein is responsible for beer foam stabilization. This report represents the first time that a brewing yeast foaming gene has been cloned and its action fully characterized.

Scoring of predicted GRK2 phosphorylation sites in Nedd4-2

MOTIVATION Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2. RESULTS We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest. CONTACT jarthur@med.usyd.edu.au

Considerations on bacterial nucleoids

The classic genome organization of the bacterial chromosome is normally envisaged with all its genetic markers linked, thus forming a closed genetic circle of duplex stranded DNA (dsDNA) and several proteins in what it is called as “the bacterial nucleoid.” This structure may be more or less corrugated depending on the physiological state of the bacterium (i.e., resting state or active growth) and is not surrounded by a double membrane as in eukayotic cells. The universality of the closed circle model in bacteria is however slowly changing, as new data emerge in different bacterial groups such as in Planctomycetes and related microorganisms, species of Borrelia, Streptomyces, Agrobacterium, or Phytoplasma. In these and possibly other microorganisms, the existence of complex formations of intracellular membranes or linear chromosomes is typical; all of these situations contributing to weakening the current cellular organization paradigm, i.e., prokaryotic vs eukaryotic cells.

Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach

BackgroundThe haemotropic mycoplasmas Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum cause feline infectious anaemia with infection rates in feline populations reflecting widespread subclinical infection. Clinically significant infections are much rarer but can be life-threatening. Current diagnosis is dependent upon visualising organisms in stained blood smears, PCR or quantitative PCR (qPCR). These procedures are labour-intensive and time-consuming. Furthermore, PCR-based approaches offer limited insight into the disease burden of the infected animal.MethodsWe have developed a novel and rapid flow cytometric system that permits diagnosis of haemotropic mycoplasma infections and quantitation of the percentage of erythrocytes that are parasitized. The method exploits the fact that mature mammalian erythrocytes, the host cell for haemoplasmas, are enucleated and thus lack nucleic acid. DRAQ5 is a synthetic anthrocycline dye which rapidly crosses cell membranes and binds to nucleic acids. The presence of exogenous bacterial DNA in mammalian erythrocytes can, therefore, be detected by DRAQ5 uptake and flow cytometric detection of DRAQ5 fluorescence.ResultsHere, we show that this system can detect epi-erythrocytic infection of companion felines by haemotropic mycoplasma. Due to their differences in size, and hence the quantity of DNA, the two major feline hemoplasmas M. haemofelis and Candidatus M. haemominutum can be distinguished according to DRAQ5 fluorescence. We have also shown the usefulness of DRAQ5 uptake in monitoring a cat infected with M. haemofelis sequentially during treatment with doxycycline.ConclusionsThe technique described is the first report of a flow cytometric method for detecting haemotropic mycoplasmas in any species and could be applied to widespread screening of animal populations to assess infection by these epi-erythrocytic parasites.