EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence

EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.     

Growth in species of the genus Ascobolus. II. (Pezizales-Ascomycota)

The kinetics of growth of six heterothallic species of the genus Ascobolus was studied in liquid culture media. The results obtained showed variation among the species in the duration of the different phases of the growth cycle. Four groups can be recognized considering the extension of the exponential phase of growth. The stationary phase, which differs in its length, is frequently very short, entering quickly in the phase of death, accompanied by the autolysis of the mycelium.     

RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.     

Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice

Duchenne muscular dystrophy is a musculoskeletal disease caused by mutations in the dystrophin gene. The purpose of this study was to use the mouse model of muscular dystrophy (mdx) to determine if the progression of the dystrophic phenotype in the diaphragm (costal) versus limb skeletal muscle (tibialis anterior) is associated with specific changes in extracellular regulated kinase (ERK1/2), p70 S6 kinase (p70(S6k)), or p38 signaling pathways. The studies detected that consistent with an earlier dystrophic phenotype, phosphorylation of p70(S6k) is elevated by 40% in the diaphragm with no change in limb muscle. In addition, phosphorylation of p38 kinase was decreased by 33% in the mdx diaphragm muscle. Levels of ERK1/2 as well as phosphorylation states were elevated in the diaphragm and limb muscle of mdx mice compared with age-matched control muscles. These results indicate that distinct signaling pathways are differentially activated in skeletal muscle of mdx mice. The specificity of these responses, particularly in the diaphragm, provides insight for potential targets for blunting the progression of the muscular dystrophy phenotype.     

Segmental identity and cerebellar granule cell induction in rhombomere 1

Background  

Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?     

Functional assessment of cryopreserved human aortas for pharmaceutical research

We evaluated the impact of standard cryopreservation on functional properties of human aortic homografts. From seven human donors, the thoracic descending aorta was obtained. Effects of cryopreservation on contractibility and endothelium function were tested. After cryopreservation no endothelium-dependent or endothelium-independent relaxation was found and the contractibility was strongly affected. Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing.     

Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases

To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli, and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of beta-lactam antibiotics.     

Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella

Background  

Sperm protein 17 (Sp17) is a three-domain protein that contains: 1) a highly conserved N-terminal domain that is 45% identical to the human type II alpha regulatory subunit (RII alpha) of protein kinase A (PKA); 2) a central sulphated carbohydrate-binding domain; and 3) a C-terminal Ca++/calmodulin (CaM) binding domain. Although Sp17 was originally discovered and characterized in spermatozoa, its mRNA has now been found in a variety of normal mouse and human tissues. However, Sp17 protein is found predominantly in spermatozoa, cilia and human neoplastic cell lines. This study demonstrates that Sp17 from spermatozoa binds A-kinase anchoring protein 3 (AKAP3), confirming the functionality of the N-terminal domain.