Activity of native hydrolytic enzymes and their association with the cell wall of three ectomycorrhizal fungi

The ecological and biogeochemical relevance of hydrolytic enzymes associated with the fungal cell wall has been poorly studied in ectomycorrhizal (ECM) fungi. We used a modified sequential extraction procedure to investigate the activity of various hydrolytic enzymes (β-glucosidase, acid-phosphatase, leucine-aminopeptidase, chitinase, xylanase and glucuronidase) and their association with the cell wall of three ECM fungi (Rhizopogon roseolus, Paxillus involutus and Piloderma croceum). Fungi were grown on C-rich solid medium under three different P concentrations (3.7, 0.37 and 0.037 mM). The sequential extraction procedure classifies enzymes as: (a) cytosolic, (b) loosely bound, (c) hydrophobically bound, (d) ionically bound and (e) covalently bound. Results showed that for the same fungus absolute enzymatic activity was affected by P concentration, whilst enzymatic compartmentalization among the cytosol and the cell wall fractions was not. The association of enzymes with the cell wall was fungus- and enzyme-specific. Our data indicate also that enzymes best known for being either extracellular or cytosolic or both, do act in muro as well. The ecological implications of cell wall-bound enzymes and the potential applications and limitations of sequential extractions are further discussed.     

Identification of Candida albicans heat shock proteins and Candida glabrata and Candida krusei enolases involved in the response to oxidative stress

In the past two decades, Candida species have become the second leading cause of invasive mycosis in immunocompromised patients. In order to colonize their hosts, these microorganisms express adhesins and cell wall proteins that allow them to adhere and neutralize the reactive oxygen species produced by phagocytic cells during the respiratory burst. However, the precise mechanism by which Candida cell wall proteins change their expression in response to oxidative stress has not been described. In an attempt to understand this change in response to oxidative stress, in this study, three Candida species, namely, C. albicans, C. glabrata and C. krusei, were exposed to increasing concentrations of H₂O₂ and induced cell wall proteins were identified by two-dimensional gel electrophoresis and peptide mass fingerprinting. Sequence analysis of differential proteins led to the identification of two heat-shock proteins in C. albicans, two enolases in C. glabrata and one enolase in C. krusei. Enolases may be involved in the protection of pathogenic cells against oxidative stress as suggested by the decrease in their expression when they were exposed to high concentrations of H₂O₂. To our knowledge, this is the first demonstration that expression of these proteins changes in response to oxidative stress in different Candida species. This knowledge can eventually facilitate both an early diagnosis and a more efficient treatment of this mycosis.     

Mixed-dimensional multi-scale poroelastic modeling of adipose tissue for subcutaneous injection

Biomechanics and Modeling in Mechanobiology - Subcutaneous injection of therapeutic monoclonal antibodies (mAbs) has gained increasing interest in the pharmaceutical industry. The transport,...     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.     

Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.     

The Notch ligand Jagged-1 is able to induce maturation of monocyte-derived human dendritic cells

Notch receptors play a key role in several cellular processes including differentiation, proliferation, and apoptosis. This study investigated whether the activation of Notch signaling would affect the maturation of dendritic cells (DCs). Direct stimulation of Notch signaling in DCs with a peptide ligand induced DC maturation, similar to LPS: DCs up-regulated maturation markers, produced IL-12, lost endocytosis capacity, and became able to activate allogeneic T cells. Furthermore, coculture of DCs with cells expressing Notch ligand Jagged-1 induced up-regulation of maturation markers, IL-12 production, T cell proliferative responses, and IFN-gamma production. Our data suggest that activation of Notch by Jagged-1 plays an important role in maturation of human DCs. Additionally, they reveal a novel role for Notch signaling in cell maturation events distal to the cell fate decision fork. These data may have important medical implications, since they provide new reagents to induce DC activity, which may be beneficial as adjuvants in situations where an immune response needs to be elicited, such as tumor immunotherapy.     

Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.