The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.     

The M4 insulator,the TM2 matrix attachment region,and the double copy of the heavy chain gene contribute to the enhanced accumulation of the PHB-01 antibody in tobacco plants

Transgenic Research - The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human...     

Quantitation of fatty acid ethyl esters in human meconium by an improved liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an improved assay for quantitation of fatty acid ethyl esters (FAEEs) in human meconium using liquid chromatography/tandem mass spectrometry (LC–MS/MS). FAAEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) and the internal standard (I.S.), ethyl heptadecanoate, were separated by reverse phase HPLC and quantified by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ionization mode. The absolute recovery of FAEEs varied from 55 ± 10% for 0.33 nmol/g (100 ng/g) of ethyl linoleate up to 86 ± 8% for 1.55 nmol/g (500 ng/g) of ethyl miristate. The LODs and LOQs varied from 0.01 to 0.08 nmol/g and from 0.02 to 0.27 nmol/g, respectively. The assay has been successfully applied to examine the FAEE levels in 81 meconium samples from babies born to mothers reporting alcohol consumption, to varying degrees, during pregnancy.     

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins,pore-forming proteins from a sea anemone

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure–function relationship, the protein–membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin–membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein–lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts–cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Sts-based vaccine platforms.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope

Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.