Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis.

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2'-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.

PTP1B dephosphorylates N-ethylmaleimide-sensitive factor and elicits SNARE complex disassembly during human sperm exocytosis.

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Bacteriostatic action of synthetic polyhydroxylated chalcones against Escherichia coli.

In previous work the bacteriostatic action of trihydroxylated chalcones against Staphylococcus aureus ATCC 25 923 was investigated. In this work the action of 2',4',2-(OH)3-chalcone, 2',4',3-(OH)3-chalcone and 2',4',4-(OH)3-chalcone against Escherichia coli ATCC 25 922 was evaluated. Growth kinetic curves of E. coli were made in nutritive broth added with increasing drug concentrations. The specific growth rates of the microorganisms were calculated by a kinetic turbidimetric method, which was previously probed and the minimal inhibitory concentrations (MIC's) were evaluated by a mechanism of action proposed. The MICs of 2',4',3-(OH)3-chalcone and 2',4',2-(OH)3-chalcone were 46 microg/ml and 122 microg/ml, respectively. The 2',4',4-(OH)3-chalcone was inactive. The MIC value of 2',4',3-(OH)3-chalcone (46 microg/ml), more active than 2',3-(OH)2-chalcone (72.2 microg/ml) may be due to the introduction of an electron donating group (-OH) at position 4' in the aromatic A-ring, which activates the region that includes the 2'-hydroxyl neighbor group and the alpha,beta-unsaturated carbonyl group.

Bacteriostatic action of synthetic polyhydroxylated chalcones against Escherichia coli.

In previous work the bacteriostatic action of trihydroxylated chalcones against Staphylococcus aureus ATCC 25 923 was investigated. In this work the action of 2,4,2-(OH)3-chalcone, 2,4,3-(OH)3-chalcone and 2,4,4-(OH)3-chalcone against Escherichia coli ATCC 25 922 was evaluated. Growth kinetic curves of E. coli were made in nutritive broth added with increasing drug concentrations. The specific growth rates of the microorganisms were calculated by a kinetic turbidimetric method, which was previously probed and the minimal inhibitory concentrations (MICs) were evaluated by a mechanism of action proposed. The MICs of 2,4,3-(OH)3-chalcone and 2,4,2-(OH)3-chalcone were 46 microg/ml and 122 microg/ml, respectively. The 2,4,4-(OH)3-chalcone was inactive. The MIC value of 2,4,3-(OH)3-chalcone (46 microg/ml), more active than 2,3-(OH)2-chalcone (72.2 microg/ml) may be due to the introduction of an electron donating group (-OH) at position 4 in the aromatic A-ring, which activates the region that includes the 2-hydroxyl neighbor group and the alpha,beta-unsaturated carbonyl group.

A comparative study of bacteriostatic activity of synthetic hydroxylated flavonoids

Among other properties, flavonoids present a notable bacteriostatic activity. In this paper, minimal inhibitory concentrations (MICs) of 5,7,4'-trihydroxyflavanone (naringenin), 5,7-dihydroxyflavone and 2',4',4- trihydroxychalcone (isoliquitirigenin) against Staphylococcus aureus ATCC 25 923 were determined and compared to values obtained for other chalcones and flavanones previously investigated. Specific growth rates and MICs were determined by a turbidimetric kinetic method. The observed sequence MICflavanone (inactive) >MIC7-hidroxyflavanone (197.6 µgml-1)>MIC5,7,4'-trihydroxyflavanone (120 µgml-1) showed that the introduction of an electron donating group (-OH) causes an increase in bioactivity. On the other hand, the comparisons MIC5,7,4'-trihydroxyflavanone (120 µgml-1) >>> MIC2',4',4-trihydroxychalcone (29 µgml-1) and MIC5,7-dihydroxyflavone (105 µgml-1) >>> MIC2',4'-dihydroxychalcone (28.8 µgml-1) indicated that the chalcone structure is the most favourable for bacteriostatic activity within the flavonoid family.