RESUMO
Molecular profiles of adhesion secretions of Gromphadorrhina portentosa (Madagascar hissing cockroach, Blattodea) were investigated by gas chromatography mass spectrometry with particular focus on a comprehensive analysis of linear and branched hydrocarbons. For this purpose, secretions from the tarsi (feet), possibly contributing to adhesion on smooth surfaces, and control samples taken from the tibiae (lower legs), which contain general cuticular hydrocarbons that are supposed to be not involved in the biological adhesion function, were analyzed and their molecular fingerprints compared. A major analytical difficulty in such a study constitutes the representative, spatially controlled, precise and reproducible sampling from a living insect as well as the minute quantities of insect secretions on both tarsi and tibiae. Thus, three different in vivo sampling methods were compared in terms of sampling reproducibility and extraction efficiency by replicate measurement of samples from tarsi and tibiae. While contact solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS) fiber showed higher peak intensities, a self-made uncoated glass fiber had the best repeatability in contact-SPME sampling. Chromatographic profiles of these two contact-SPME sampling methods were statistically not significantly different. Inter-individual variances were larger than potentially existing minor differences in molecular patterns of distinct sampling methods. Sampling by solvent extraction was time consuming, showed lower sensitivities and was less reproducible. In general, sampling by contact-SPME with a cheap glass fiber turned out to be a viable alternative to PDMS-SPME sampling. Hydrocarbon patterns of the tarsal adhesion secretions were qualitatively similar to those of epicuticular hydrocarbon profiles of the tibiae. However, hydrocarbons were in general less abundant in tarsal secretions than secretions from tibiae.
Assuntos
Adesão Celular/fisiologia , Dimetilpolisiloxanos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Vidro/química , Hidrocarbonetos/análise , Microextração em Fase Sólida/métodos , Solventes/química , Animais , Baratas , Madagáscar , Reprodutibilidade dos Testes , Tíbia/metabolismoRESUMO
Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.
Assuntos
Antimaláricos , Inibidores Enzimáticos , Quelantes de Ferro , Oxigenases de Função Mista/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/química , Antimaláricos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Quelantes de Ferro/química , Quelantes de Ferro/farmacologiaRESUMO
This article reports on the chemical analysis of molecular profiles of tarsal secretions of the desert locust Schistocerca gregaria (Forsskål, 1775) by gas chromatography hyphenated with quadrupol mass spectrometry (GC-MS) as well as (1)H-nuclear magnetic resonance ((1)H NMR) spectroscopy. Special focus of this study was to elaborate on sampling methods which enable selective microscale extraction of insect secretions in a spatially controlled manner, in particular tarsal adhesive secretions and secretions located on cuticle surfaces at the tibia. Various solvent sampling procedures and contact solid-phase microextraction (SPME) methods were compared in terms of comprehensiveness and extraction efficiencies as measured by signal intensities in GC-MS. Solvent sampling with water as extraction solvent gave access to the elucidation of chemical profiles of polar compound classes such as amino acids and carbohydrates, but is extremely tedious. Contact SPME on the other hand can be regarded as a simplified and more elegant alternative, in particular for the lipophilic compound fraction. Many proteinogenic amino acids and ornithine as well as carbohydrate monomers arabinose, xylose, glucose, and galactose were detected in tarsal secretions after acid hydrolysis of aqueous extracts. Qualitatively similar but quantitatively significantly different molecular profiles were found for the lipid fraction which contained mainly n-alkanes and internally branched monomethyl-, dimethyl-, and trimethyl-alkanes in the C23-C49 range as well as long chain fatty acids and aldehydes. Especially, hydrocarbons with >C40 carbon numbers have previously been rarely reported for insect secretions. The results suggest that the investigated insect secretions are complex emulsions which allow the attachment of tarsi on various otherwise incompatible materials of smooth and rough surfaces. The solid consistence of the established alkanes at ambient temperatures might contribute to a semi-solid consistence of the adhesive, amalgamating partly opposing functions such as slip resistance, tarsal release, desiccation resistance, and mechanical compliance. The methods developed can be extended to other similar applications of studying compositions of insect secretions of other species.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Gafanhotos/metabolismo , Animais , Microextração em Fase SólidaRESUMO
East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy.