The role of amino acid residues in the active site of a midgut microvillar aminopeptidase from the beetle Tenebrio molitor

Aminopeptidases are major enzymes in the midgut microvillar membranes of most insects and are targets of insecticidal Bacillus thuringiensis crystal delta-endotoxins. Sequence analysis and substrate specificity studies showed that these enzymes resemble mammalian aminopeptidase N, although information on the organization of their active site is lacking. The effect of pH at different temperatures on the kinetic parameters of Tenebrio molitor (Coleoptera) larval aminopeptidase showed that enzyme catalysis depend on a deprotonated (pK 7.6; DeltaH degrees (ion), 7.6 kJ/mol) and a protonated (pK 8.2; DeltaH degrees (ion), 16.8 kJ/mol) group. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and diethylpyrocarbonate inactivate the enzyme by modifying a pK 5.8 carboxylate and a imidazole group, respectively, with a reaction order around 1. Tetranitromethane changes the K(m) of the enzyme without affecting its V(max) by modifying a phenol group. The presence of a competitive inhibitor decrease the inactivation reaction rates in all these cases. EDTA inactivation of the aminopeptidase is affected by pH and temperature suggesting the involvement in metal binding of at least one deprotonated imidazole group (pK 5.8, DeltaH degrees (ion), 20 kJ/mol). The data support the hypothesis that T. molitor aminopeptidase catalysis depends on a catalytic metal and on a carboxylate and a protonated imidazole group, whereas substrate binding relies in one phenol and one carboxylate groups. The insect aminopeptidase shares common features with mammalian aminopeptidase N, although differing in details of substrate binding and in residues directly involved in catalysis.     

The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547-1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous "precentrioles" become morphologically recognizable centrioles before mitosis. De novo-assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo-formed centrioles do not mature if they are assembled in S phase-arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.     

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: bruchidae) and the induction of alpha-amylases in response to different diets

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.     

Lysophosphatidic acid as an autocrine and paracrine mediator

Recent studies have established that lysophosphatidic acid (LPA) is produced by a wide variety of cell types, and that most mammalian cells express receptors for LPA. These findings raise the hypothesis that LPA acts as an autocrine mediator to initiate signaling in the cells where it is produced, as well as a paracrine mediator to affect neighboring cells. The extent to which these scenarios occur will depend on the species of LPA generated, the LPA receptors expressed, and the ability of these receptors to bind to the LPA produced. The enzymes involved in LPA synthesis and their cellular localization in relationship to LPA receptors are also likely to be important. Studies addressing these issues with respect to the potential roles of LPA as an autocrine and paracrine mediator are reviewed, with examples.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

Craft Beers Fermented by Potential Probiotic Yeast or Lacticaseibacilli Strains Promote Antidepressant-Like Behavior in Swiss Webster Mice

Probiotics and Antimicrobial Proteins - This study aimed to produce a probiotic-containing functional wheat beer (PWB) by an axenic culture system with potential probiotic Saccharomyces cerevisiae...     

Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes

The way specific procyanidins exert their anti-inflammatory effects is not fully understood. This study has investigated the capacity of different procyanidins to modulate lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production in THP1 human monocytes and their effects on the redox regulated protein kinases activity: IkB kinase beta (IKKb) and the extracellular signal-regulated kinase (ERK). LPS-triggered increase of ROS was prevented by cell pre-incubation with procyanidins. LPS induced ERK1/2 activation through phosphorylation, which was inhibited by all the compounds tested, the most active being epigallocatechin (EG), followed by epigallocatechin gallate (EGCG) and C1. Procyanidins inhibited IKKb activity in vitro. C1 and procyanidin extract (PE) exerted the maximal IKKb inhibition, followed by EGCG and dimer B1. Catechin exerted a slight but significant IKKb inhibition, in contrast to epicatechin, which was ineffective. In conclusion, procyanidins reduce the LPS-induced production of ROS and they exert their anti-inflammatory effects by inhibiting ERK1/2 and IKKb activity.     

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106kDa (gel filtration) and 55kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, AβNA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3, Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species.     

Amphiphilic amylose-g-poly(meth)acrylate copolymers through "click" onto grafting method

Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(α)- or end-(ω)-azide functionalized poly(meth)acrylates to be used as "click" reagents in Cu(I) catalyzed azide-alkyne [3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA). Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.     

Impact of the California lead ammunition ban on reducing lead exposure in golden eagles and turkey vultures

Predatory and scavenging birds may be exposed to high levels of lead when they ingest shot or bullet fragments embedded in the tissues of animals injured or killed with lead ammunition. Lead poisoning was a contributing factor in the decline of the endangered California condor population in the 1980s, and remains one of the primary factors threatening species recovery. In response to this threat, a ban on the use of lead ammunition for most hunting activities in the range of the condor in California was implemented in 2008. Monitoring of lead exposure in predatory and scavenging birds is essential for assessing the effectiveness of the lead ammunition ban in reducing lead exposure in these species. In this study, we assessed the effectiveness of the regulation in decreasing blood lead concentration in two avian sentinels, golden eagles and turkey vultures, within the condor range in California. We compared blood lead concentration in golden eagles and turkey vultures prior to the lead ammunition ban and one year following implementation of the ban. Lead exposure in both golden eagles and turkey vultures declined significantly post-ban. Our findings provide evidence that hunter compliance with lead ammunition regulations was sufficient to reduce lead exposure in predatory and scavenging birds at our study sites.