Intracellular damage and death caused by protease inhibitors on Alexandrium catenella natural cysts and vegetative cells

Proteases from a Chilean clone of Alexandrium catenella were studied using gelatin–zymogram gel and protease fluorescent substrate to facilitate their visual identification in vitro and in vivo, respectively. Proteolytic activity bands were grouped arbitrarily according to their molecular weight as P1 (150 and 120 kDa), P2 (100 kDa), P3 (70 and 65 kDa), P4 (60, 55 and 50 kDa) and P5 (25 kDa). Protease inhibitors affect differentially P2 and P3 proteases. Only P2 activity increased in the presence of 1–10 phenanthroline (σPhe), pepstatin A (pepA), leupeptin (leup) and phenylmethanesulfonyl fluoride (PMSF), while P2 and P3 become inactivated with ρ-aminobenzamidine. The protease inhibitors lethal dose was determined by incubating cells with different concentration of the protease inhibitor and evaluating their effect on cell viability. Furthermore, cells treated for 4 h with one lethal dose of 1–10 phenanthroline and ρ-aminobenzamidine, caused serious damage to the intracellular vacuolar system and nuclear material. Live cysts also die when treated independently with these two protease inhibitors. Future work will be aimed at chemically designing species-specific inhibitors for their potential use in killing cysts transported within the sediment of ship ballast water before washing them off to the environment.     

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO₄)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO₄) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO₄) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO₄) residues in both CS and DS. IdoA-GalNAc(4,6-SO₄) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO₄) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO₄) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.     

Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (−2713 bp) upstream of its open reading frame and three upstream-truncated fragments (−1912, −1060 and −560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at −315 and −274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2 mM menadione, and in colonies under 6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40 °C after 3-day growth at 25 °C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.     

Application of infrared thermography as an indicator of heat and methane production and its use in the study of skin temperature in response to physiological events in dairy cattle (Bos taurus)

There is a demand in the livestock industry for alternative assessments of feed efficiency. Infrared thermography was tested for predicting heat production, methane production and for the detection of physiological events (e.g. heat increment of feeding) in dairy cattle. Multiple body locations were infrared scanned concomitantly with the measurement of the animals’ gaseous exchange. Infrared thermography can be successfully applied for assessing heat and methane production, through the analysis of feet temperature and temperature difference between left and right flanks, respectively. This technology is also useful for assessing physiological responses to milking and feeding.     

Entamoeba histolytica: Effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.