Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

Characterization of the isozymes of pyruvate dehydrogenase phosphatase: implications for the regulation of pyruvate dehydrogenase activity

The activity of mammalian pyruvate dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of pyruvate dehydrogenase phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the alpha chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of pyruvate dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of pyruvate dehydrogenase.     

The composition and timing of flower odour emission by wild <Emphasis Type="Italic">Petunia axillaris</Emphasis> coincide with the antennal perception and nocturnal activity of the pollinator <Emphasis Type="Italic">Manduca sexta</Emphasis>

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.     

The Sec14 homology domain regulates the cellular distribution and transforming activity of the Rho-specific guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose overexpression cause deregulated growth in murine fibroblasts. Dbs contains multiple recognizable motifs including a centrally located Rho-specific guanine nucleotide exchange factor domain, a COOH-terminal Src homology 3 domain, two spectrin-like repeats, and a recently identified NH(2)-terminal Sec14 homology domain. The transforming potential of Dbs is substantially activated by the removal of inhibitory sequences that lie outside of the core catalytic sequences, and in this current study we mapped this inhibition to the Sec14 domain. Surprisingly removal of the NH(2) terminus did not alter the catalytic activity of Dbs in vivo but rather altered its subcellular distribution. Whereas full-length Dbs was distributed primarily in a perinuclear structure that coincides with a marker for the Golgi apparatus, removal of the Sec14 domain was associated with translocation of Dbs to the cell periphery where it accumulated within membrane ruffles and lamellipodia. However, translocation of Dbs and the concomitant changes in the actin cytoskeleton were not sufficient to fully activate Dbs transformation. The Sec14 domain also forms intramolecular contacts with the pleckstrin homology domain, and these contacts must also be relieved to achieve full transforming activity. Collectively these observations suggest that the Sec14 domain regulates Dbs transformation through at least two distinct mechanisms, neither of which appears to directly influence the in vivo exchange activity of the protein.     

The vertical distribution of phytoplankton in stratified water columns

What determines the vertical distribution of phytoplankton in different aquatic environments remains an open question. To address this question, we develop a model to explore how phytoplankton respond through growth and movement to opposing resource gradients and different mixing conditions. We assume stratification creates a well-mixed surface layer on top of a poorly mixed deep layer and nutrients are supplied from multiple depth-dependent sources. Intraspecific competition leads to a unique strategic equilibrium for phytoplankton, which allows us to classify the distinct vertical distributions that can exist. Biomass can occur as a benthic layer (BL), a deep chlorophyll maximum (DCM), or in the mixed layer (ML), or as a combination of BL+ML or DCM+ML. The ML biomass can be limited by nutrients, light, or both. We predict how the vertical distribution, relative resource limitation, and biomass of phytoplankton will change across environmental gradients. We parameterized our model to represent potentially light and phosphorus limited freshwater lakes, but the model is applicable to a broad range of vertically stratified systems. Increasing nutrient input from the sediments or to the mixed layer increases light limitation, shifts phytoplankton towards the surface, and increases total biomass. Increasing background light attenuation increases light limitation, shifts the phytoplankton towards the surface, and generally decreases total biomass. Increasing mixed layer depth increases, decreases, or has no effect on light limitation and total biomass. Our model is able to replicate the diverse vertical distributions observed in nature and explain what underlying mechanisms drive these distributions.     

Analysis of the S. pombe signalling scaffold protein Cdc11p reveals an essential role for the N-terminal domain in SIN signalling

The initiation of cytokinesis in the fission yeast Schizosaccharomyces pombe is signalled by the septation initiation network (SIN). Signalling originates from the spindle pole body (SPB), where SIN proteins are anchored by a scaffold composed of cdc11p and sid4p. Cdc11p links the other SIN proteins to sid4p and the SPB. Homologues of cdc11p have been identified in Saccharomyes cerevisiae (Nud1p) and human cells (Centriolin). We have defined functional domains of cdc11p by analysis of deletion mutants. We demonstrate that the C-terminal end of cdc11p is necessary for SPB localisation. We also show that the N-terminal domain is necessary and sufficient for signal transduction, since tethering of this domain to the SPB will substitute for cdc11p in SIN function.     

CoMFA and CoMSIA analyses on 1,2,3,4-tetrahydropyrrolo[3,4-b]indole and benzimidazole derivatives as selective CB2 receptor agonists

Novel classes of cannabinoid 2 receptor (CB2) agonists based on 1,2,3,4-tetrahydropyrrolo[3,4-b]indole and benzimidazole scaffolds have shown high binding affinity toward CB2 receptor and good selectivity over cannabinoid 1 receptor (CB1). A computational study of comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) was performed, initially on each series of agonists, and subsequently on all compounds together, in order to identify the key structural features impacting their binding affinity. The final CoMSIA model resulted to be the more predictive, showing cross-validated r² (r_cv ²) = 0.680, non cross-validated r² (r_ncv ²) = 0.97 and test set r²( r_pred² ) = 0.93 {{\hbox{r}}^2}\left( {{\hbox{r}}_{\rm{pred}}^2} \right) = 0.{93} . The study provides useful suggestions for the design of new analogues with improved affinity.     

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca²⁺ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF''s profibrotic function in the heart.