The toxicity of angiotensin converting enzyme inhibitors to larvae of the disease vectors Aedes aegypti and Anopheles gambiae

The control of mosquitoes is threatened by the appearance of insecticide resistance and therefore new control chemicals are urgently required. Here we show that inhibitors of mosquito peptidyl dipeptidase, a peptidase related to mammalian angiotensin-converting enzyme (ACE), are insecticidal to larvae of the mosquitoes, Aedes aegypti and Anopheles gambiae. ACE inhibitors (captopril, fosinopril and fosinoprilat) and two peptides (trypsin-modulating oostatic factor/TMOF and a bradykinin-potentiating peptide, BPP-12b) were all inhibitors of the larval ACE activity of both mosquitoes. Two inhibitors, captopril and fosinopril (a pro-drug ester of fosinoprilat), were tested for larvicidal activity. Within 24 h captopril had killed >90% of the early instars of both species with 3rd instars showing greater resistance. Mortality was also high within 24 h of exposure of 1st, 2nd and 3rd instars of An. gambiae to fosinopril. Fosinopril was also toxic to Ae. aegypti larvae, although the 1st instars appeared to be less susceptible to this pro-drug even after 72 h exposure. Homology models of the larval An. gambiae ACE proteins (AnoACE2 and AnoACE3) reveal structural differences compared to human ACE, suggesting that structure-based drug design offers a fruitful approach to the development of selective inhibitors of mosquito ACE enzymes as novel larvicides

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement. Electronic supplementary material The online version of this article (doi:10.1186/s12868-014-0132-4) contains supplementary material, which is available to authorized users

Transcriptome Analysis of the Desert Locust Central Nervous System: Production and Annotation of a Schistocerca gregaria EST Database

) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology

Structural diversity of angiotensin-converting enzyme : insights from structure-activity comparisons of two Drosophila enzymes.

The crystal structure of a Drosophila angiotensin-converting enzyme (ANCE) has recently been solved, revealing features important for the binding of ACE inhibitors and allowing molecular comparisons with the structure of human testicular angiotensin-converting enzyme (tACE). ACER is a second Drosophila ACE that displays both common and distinctive properties. Here we report further functional differences between ANCE and ACER and have constructed a homology model of ACER to help explain these. The model predicts a lack of the Cl–-binding sites, and therefore the strong activation of ACER activity towards enkephalinamide peptides by NaCl suggests alternative sites for Cl– binding. There is a marked difference in the electrostatic charge of the substrate channel between ANCE and ACER, which may explain why the electropositive peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low Km, but does not bind to ACER. Bradykinin (BK) peptides are excellent ANCE substrates. Models of BK docked in the substrate channel suggest that the peptide adopts an N-terminal β-turn, permitting a tight fit of the peptide in the substrate channel. This, together with ionic interactions between the guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150 in the S2' pocket, are possible reasons for the high-affinity binding of BK. The replacement of Asp360 with a histidine in ACER would explain the higher Km recorded for the hydrolysis of BK peptides by this enzyme. Other differences in the S2' site of ANCE and ACER also explain the selectivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER. These structural and enzymatic studies provide insight into the molecular basis for the distinctive enzymatic features of ANCE and ACER

Transcriptomic and Expression Analysis of the Salivary Glands in White-Backed Planthoppers, Sogatella furcifera

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the serious rice pests because of its destructive feeding. The salivary glands of the WBPH play an important role in the feeding behaviour. Currently, however, very little is known about the salivary glands at the molecular level. We sequenced the salivary gland transcriptome (sialotranscripome) of adult WBPHs using the Illumina sequencing. A total of 65,595 transcripts and 51,842 unigenes were obtained from salivary glands. According to annotations against the Nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. In the present study, we identified 32 salivary protein genes from the WBPH sialotranscripome, which were categorized as those involved in sugar metabolism, detoxification, suppression of plant defense responses, immunity-related responses, general digestion, and other phytophagy processes. Tissue expression profiles analysis revealed that four of 32 salivary protein genes (multicopper oxidase 4, multicopper oxidase 6, carboxylesterase and uridine phosphorylase 1 isform X2) were primarily expressed in the salivary gland, suggesting that they played putative role in insect-rice interactions. 13 of 32 salivary protein genes were primarily expressed in gut, which might play putative role in digestive and detoxify mechanism. Development expression profiles analysis revealed that the expression level of 26 of 32 salivary protein genes had no significant difference, suggesting that they may play roles in every developmental stages of salivary gland of WBPH. The other six genes have a high expression level in the salivary gland of adult. 31 of 32 genes (except putative acetylcholinesterase 1) have no significant difference in male and female adult, suggesting that their expression level have no difference between sexes. This report analysis of the sialotranscripome for the WBPH, and the transcriptome provides a foundational list of the genes involved in feeding. Our data will be useful to investigate the mechanisms of interaction between the WBPH and the host plant

Deorphanization of Novel Peptides and Their Receptors

Peptide hormones and neuropeptides play important roles in endocrine and neural signaling, often using G protein-coupled receptor (GPCR)-mediated signaling pathways. However, the rate of novel peptide discovery has slowed dramatically in recent years. Genomic sequencing efforts have yielded a large number of cDNA sequences that potentially encode novel candidate peptide precursors, as well as hundreds of orphan GPCRs with no known cognate ligands. The complexity of peptide signaling is further highlighted by the requirement for specific posttranslational processing steps, and these must be accomplished in vitro prior to testing newly discovered peptide precursor candidates in receptor assays. In this review, we present historic as well as current approaches to peptide discovery and GPCR deorphanization. We conclude that parallel and combinatorial discovery methods are likely to represent the most fruitful avenues for both peptide discovery as well as for matching the remaining GPCRs with their peptide ligands