Comparison of fatty acid contents and composition in major lipid classes of larvae and adults of mosquitoes (Diptera: Culicidae) from a steppe region

Emerging aquatic insects, including mosquitoes, are known to transfer to terrestrial ecosystems specific essential biochemicals, such as polyunsaturated fatty acids (PUFA). We studied fatty acid (FA) composition and contents of dominant mosquito populations (Diptera: Culicidae), that is, Anopheles messeae, Ochlerotatus caspius, Oc. flavescens, Oc. euedes, Oc. subdiversus, Oc. cataphylla, and Aedes cinereus, inhabited a steppe wetland of a temperate climate zone to fill up the gap in their lipid knowledge. The polar lipid and triacylglycerol fractions of larvae and adults were compared. In most studied mosquito species, we first found and identified a number of short‐chain PUFA, for example, prominent 14:2n‐6 and 14:3n‐3, which were not earlier documented in living organisms. These PUFA, although occurred in low levels in adult mosquitoes, can be potentially used as markers of mosquito biomass in terrestrial food webs. We hypothesize that these acids might be synthesized (or retroconverted) by the mosquitoes. Using FA trophic markers accumulated in triacylglycerols, trophic relations of the mosquitoes were accessed. The larval diet comprised green algae, cryptophytes, and dinoflagellates and provided the mosquitoes with essential n‐3 PUFA, linolenic, and eicosapentaenoic acids. As a result, both larvae and adults of the studied mosquitoes had comparatively high content of the essential PUFA. Comparison of FA proportions in polar lipids versus storage lipids shown that during mosquito metamorphosis transfer of essential eicosapentaenoic and arachidonic acids from the reserve in storage lipids of larvae to functional polar lipids in adults occurred.     

Physiological and anatomical disturbances induced by chloride salts in sensitive and tolerant citrus: beneficial and detrimental effects of cations

In citrus, the relative contributions of chloride and cations to growth disturbances induced by salinity are a matter of controversy. Chloride salts (15 mol m^–3 CaCl₂, 30 mol m^–3 CaCl and 30 mol m^–3 KCl) reduced growth and gas exchange parameters, increased leaf damage and abscission and produced anatomical disarrangements and mineral imbalances in seedlings of sensitive Carrizo citrange ( Citrus sinensis x Poncirus trifoliata ) and tolerant Cleopatra mandarin ( Citrus reshni ). In both cultivars, Ca²⁺ was more beneficial, and K⁺ more detrimental, for growth than sodium. Photosynthesis and growth disturbances were highly correlated ( P ≤ 0·001) with leaf Cl^– build-up. In the sensitive genotype, Cl^– was also significantly correlated with several leaf anatomical disarrangements, such as increase in succulence. In comparison with sodium, both calcium and potassium increased leaf Cl^– content (up to 25 and 69%, respectively). Protective calcium effects were not linked to improvement of photosynthesis, reduction of leaf anatomical disarrangements, or prevention of Cl^– and Na⁺ increases. It is proposed that the ameliorative effects of calcium on citrus grown under salinity are mostly related to reduction of leaf abscission. Collectively, the data suggest a cause–effect relationship between Cl^– build-up and reduced growth, whereas chloride correlations with declines in photosynthesis or increases in succulence appear to be indirect.     

Antibacterial effect of honey on the in vitro and in vivo growth of Escherichia coli

The study investigated the antibacterial effect of honey against pathogenic Escherichia coli. Honey showed inhibitory activity against the growth of E. coli (ATCC 25922) in agar plate assay. In liquid culture (48 h, 37 °C) the growth rate of bacterial cells decreased in the presence of honey (9.6 × 10⁵ c.f.u./ml) compared with sucrose (2.87 × 10⁸ c.f.u./ml). Rats fed with honey and orally inoculated with E. coli excreted significantly (P < 0.05) less bacterial cells in faeces compared to controls. Animals acclimatized to feeding of honey prior to E. coli inoculation showed a significant decrease in excreted bacterial load compared with the group provided with honey after bacterial inoculation. Consumption of honey also enhanced the concentration of short chain fatty acids in the intestine of rats (83 mM) compared with the control group (44.5 mM). The results show that honey possessed significant antibacterial activity against E. coli under in vitro and in vivo conditions, and indicate the potential benefit of consumption of honey regularly on the microbiological constitution of animals feeding on it.     

A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies

Several studies have shown that the fatty acid composition of circulating lipids reflects dietary fat intake, in turn being related to health status. The fatty acid composition of plasma lipids is therefore an important parameter in studies on dietary interventions. The aim of our study was to develop a rapid and inexpensive method for the analysis of circulating fatty acids applicable to large population groups. Drops of blood collected from fingertips have been directly subjected to transmethylation for gas chromatography analysis. This new method, validated for reproducibility, has been compared with the conventional method, based on withdrawal of blood from the antecubital vein followed by lipid extraction, and identical data have been obtained with the two techniques. Observed and predicted differences between blood and plasma fatty acids are related to the contribution of circulating cell membranes in blood. Finally the application of the methods to samples from 100 healthy subjects and the assessed correlation between dietary habits and blood fatty acid profiles demonstrate the validity of the new method and its applicability to nutritional and epidemiological studies.     

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.     

Paramagnetic probes in NMR and EPR studies of membrane enzymes

The applications of paramagnetic probes to problems of structure and mechanism are discussed from the point of view of the membrane enzymologist. Problems unique to membrane systems are discussed, and a variety of nuclear and paramagnetic probes are evaluated. Three membrane ATPase (kidney (Na+ + K+)-ATPase, Ca2+-ATPase from sarcoplasmic reticulum and Mg2+-ATPase from kidney) are used to describe the types of experiments which can be done, the information which can be obtained and the limitations involved. Nuclear relaxation studies employing 1H, 7Li+, 31P and 205Tl+ nuclei are described. The advantages and disadvantages of Mn2+, Gd3+ and Cr3+ as paramagnetic probes are discussed in terms of the three ATPases. The theory and interpretation of Mn2+ and Gd3+ EPR spectra are evaluated in studies with the (Na+ + K+)-ATPase and Ca2+-ATPase, respectively.     

Uninfected red cells from malaria-infected blood: Alteration of fatty acid composition involving a serum protein: An in vivo and in vitro study

Summary Alteration of uninfected erythrocytes, fromPlasmodium (the malaria parasite)-infected blood remained an open question. In this study we compared the in vivo fatty acid compositions of control and uninfected monkey erythrocytes. A large (40%) increase in the linoleic acid level was observed, which was recovered mostly in neutral lipids. An in vitro system was developed to study medium-mediated alterations of cultured erythrocytes byPlasmodium falciparum. The increase in the linoleate level was reproduced in vitro and was also localized in the neutral lipid fraction, especially in triacylglycerols. Studies using proteolytic digestion and heat denaturation showed that a heat-labile serum protein is indispensable for the increase in the linoleate level of red cells treated with the supernatant, ofP. falciparum cultures. Both the function and the mechanism of this modification of uninfected erythrocytes still remain unknown. This work was supported by the UNDP/World Bank/WHO special program for Research and Training in Tropical Diseases (grant T16-181-M2-15B).     

Growth and ion accumulation of two rapid-cycling Brassica species differing in salt tolerance

The response of two rapid-cycling Brassica species differing in tolerance to seawater salinity was studied over a period of 24 days. In response to 8 dS m⁻¹ salinity, the two Brassica species showed clear differences in the changes in relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR). The RGR of B. napus was slightly reduced by salinity, wheareas the RGR of B. carinata was largely reduced in the early stages of salinization. LAR of B. napus was affected by salinity in the later stages of growth and significantly correlated with the reduction in RGR. On the other hand, the NAR of B. carinata was decreased by salinity, corresponding to the decrease of the RGR of B. carinata. The NAR of B. napus was not significantly affected by salinity according to analysis of covariance. The shoot concentrations of Na, Mg and Cl increased while the concentrations of K and Ca decreased sharply during the first 5 days of salinization; subsequently, all ion concentrations remained relatively constant. The concentrations of Na, K, Ca, Mg and Cl in the root were similarly affected by salinity. There were no significant differences of ion concentrations between species that could be related to the differences in salt tolerance. Thus, the differences in salt tolerance between species can not be related to differences in specific-ion effects, but may be related to some factor that reduces the NAR of B. carinata during the early stages of growth.     

Protective effects of Brussels sprouts, oligosaccharides and fermented milk towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in the human flora associated F344 rat: role of xenobiotic metabolising enzymes and intestinal microflora

We investigated the chemoprotective effects of four common constituents of the human diet, i.e. a fermented milk, inulin, oligofructose and Brussels sprouts, towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in male Fischer 344 rats harbouring a human intestinal microflora. We found that the four dietary components significantly reduced IQ-induced DNA damage in hepatocytes (reduction ranged from 74% with inulin to 39% with Brussels sprouts) and colonocytes (reduction ranged from 68% with inulin to 56% with Brussels sprouts). This chemoprotective effect correlated with the induction of hepatic UDP-glucuronosyl transferase following Brussels sprouts consumption, and with alterations of bacterial metabolism in the distal gut (acidification, increase of butyrate proportion, decrease of beta-glucuronidase activity) following inulin consumption.     

Combined effect of vanadium(V) and chromium(III) on lipid peroxidation in liver and kidney of rats

Since chromium(III) was demonstrated to have antioxidative action, we have decided to study the effect of this element on V-induced LPO in liver and kidney of rats. Outbred 2-month-old, albino male Wistar rats received daily, for a period of 12 weeks: group I (control), deionized water to drink; group II, sodium metavanadate (SMV) solution at a concentration of 0.100mgV/mL; group III, chromium chloride (CC) solution at a concentration of 0.004mgCr/mL and group IV, SMV-CC solution at a concentration of 0.100mgV and 0.004mgCr/mL. The particular experimental groups took up with drinking water about 8.6mgV/kg b.w./24h (group II), 0.4mgCr/kg b.w./24h (group III), 9mgV and 0.36mgCr/kg b.w./24h (group IV). The V- or Cr-treated groups had higher concentrations of these two elements in liver and kidney compared to the controls. The administration of vanadium alone caused a significant decrease in fluid intake and in body weight gain compared to the controls. In liver supernatants obtained from all tested rats a statistically significant increase in MDA concentration was demonstrated in spontaneous LPO in comparison with the control rats. Moreover, in rats intoxicated with vanadium alone a statistically significant increase in liver MDA level was observed in the presence of 100microM NaVO(3). Instead, in supernatants of liver received from rats treated with chromium alone, a statistically significant increase in MDA concentration in comparison with the controls was found in the presence of 400microM NaVO(3). In kidney supernatants obtained from rats treated with chromium alone, a statistically significant increase in lipid peroxidation was shown in the presence of 30microM FeSO(4) and 400microM NaVO(3). These results show that the tested doses of vanadium(V) and chromium(III) ingested by rats with their drinking water caused significant alterations in internal organs, especially in liver. Under the conditions of our experiment, Cr(III) did not demonstrate antioxidant action, it rather had an oxidant effect.     

Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis

The three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol^− 1. This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity

The growth of six rapid-cycling lines of Brassica species, B. napus, B. campestris, B. nigra, B. juncea, B. oleracea and B. carinata was inhibited by seawater salinity. Based on the change in dry matter reduction relative to the control at varying concentrations of salts (4, 8 and 12 dS m^-1), the relative salt tolerance of these species was evaluated. B. napus and B. carinata were the most tolerant and most sensitive species, respectively, while the other four species were moderately tolerant. The influence of seawater on the concentrations of 12 elements including macronutrients and micronutrients in the shoots of these Brassica plants was characterized to determine the relationship between nutritional disturbance and relative salt tolerance. It was found that seawater salinity had a significant effect on the concentrations of Ca, Mg, K, Cl, Na and total N in the shoots of these plants but only the change in Ca concentration was significantly related to the relative salt tolerance of these six rapid-cycling Brassica species according to a rank analysis of the data. This finding indicates that Ca may play a regulatory role in the responses of Brassica species to saline conditions.     

Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2)

The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.     

Identification of a novel large intragenic deletion in a family with Fanconi anemia: First molecular report from India and review of literature

We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature, hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2 Western blot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel homozygous large intragenic deletion (exons 8–27 del) in the FANCA gene in the proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation. We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different countries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is the first molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene. Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper genetic counseling to the family.     

Ca mishandling and cardiac dysfunction in obesity and insulin resistance: Role of oxidative stress

Obesity and insulin resistance (IR) are strongly connected to the development of subclinical cardiac dysfunction and eventually can lead to heart failure, which is the main cause of morbidity and death in patients having these metabolic diseases. It has been considered that excessive fat tissue may play a critical role in producing systemic IR and enhancing reactive oxygen species (ROS) generation. This oxidative stress (OS) may elicit or exacerbate IR. On the other hand, evidence suggests that some of the cellular mechanisms involved in the pathophysiology of obesity and IR-related cardiomyopathy are excessive myocardial ROS production and abnormal Ca²⁺ homeostasis. In addition, emerging evidence suggests that augmented ROS production may contribute to Ca²⁺ mishandling by affecting the redox state of key proteins implicated in this process. In this review, we focus on the role of Ca²⁺ mishandling in the development of cardiac dysfunction in obesity and IR and address the evidence suggesting that OS might also contribute to cardiac dysfunction by affecting Ca²⁺ handling.     

Functional genetic variation in aminopeptidase A (ENPEP): lack of clear association with focal and segmental glomerulosclerosis (FSGS)

The aminopeptidase A (APA) ectopeptidase is an integral membrane-bound zinc metalloprotease that cleaves aspartic and glutamic acidic residues from the N-terminus of a number of protein substrates that includes angiotensin II. Angiotensin II, the most vasoactive component of the renin-angiotensin-aldosterone (RAAS) pathway, can contribute to renal disease by causing an increase in arterial blood pressure leading to glomerular injury and fibrosis. APA is expressed in many organs, including the kidney where it localizes mainly to the podocyte cell membrane and brush borders of the proximal tubule cells. Antibodies directed to the APA peptide can induce an acute massive albuminuria in wild-type BALB/c mice after intravenous injection. We examined whether variants in the APA encoding gene (ENPEP) are more frequent in individuals with the proteinuric disease focal and segmental glomerulosclerosis (FSGS) compared to control individuals. The ENPEP coding sequence was re-sequenced in 188 FSGS patients and 48 controls. Genetic variants were further genotyped in 181 individuals without any known kidney disease. We then examined the effect of the non-synonymous coding variants identified on their cell surface APA activity after transfection in COS-1 cells. Several of these ENPEP variants lead to reproducibly altered APA activity. However, we did not see a clear correlation between the presence of a functional ENPEP variant and FSGS. However, the existence of these variants with marked effect on APA activity suggests that both rare and common variation in ENPEP may contribute to the development of renal and hypertensive disorders and warrants further study.     

Intra-specific hybridization: generator of genetic diversification and heterosis in Andrographis paniculata Nees. A bridge from extinction to survival

Andrographis paniculata (AP) has been stated as a low-diverse, endangered and red-listed plant species. Self-pollinated mating system, being an introduced species and experiencing a bottleneck as well as over exploitation cause such a consequence. Inter and intra-specific hybridizations have been suggested as essential techniques for generating genetic diversity. To test the effect of intra-specific hybridization on diversification and heterosis of AP, seven accessions were outcrossed manually in all 21 possible combinations. Three types of markers including morphological, phytochemical and RAPD markers were employed to evaluate the mentioned hypothesis. The results revealed that hybridization acted as a powerful engine for diversification of AP as it caused heterotic expression of the studied traits, simultaneously. Initially, it seems that additive and non-additive gene effects both can be considered as the genetic basis of heterosis in AP for the investigated traits. Agronomic and morphological traits were differentiated from each other, while positive heterosis was recorded mainly for agronomic traits but not for the morphological traits. Intra-specific hybridization increased the genetic diversity in AP population. Nevertheless, a part of this variation could also be attributed to the negative heterosis. The current exploration demonstrated the first ever conducted manual intra-specific hybridization among AP accessions in a mass scale. However, the 17 RAPD primers produced a monomorph pattern, but perhaps increasing the number of markers can feature a new genetic profile in this plant.