Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Biologically active polypeptides of sea anemones: Structure, function, and prospects for application

This paper presents the results of a structure-functional study of neurotoxins, actinoporins, and proteinase inhibitors from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus) and the low boreal sea anemone Oulactis orientalis. The new data significantly contribute to the knowledge of the mechanisms of action of polypeptides from marine coelenterates and can be useful for solving practical problems of the biotechnological development of medicines.     

A serine protease inhibitor from the anemone <Emphasis Type="Italic">Radianthus macrodactylus</Emphasis>: Isolation and physicochemical characteristics

A serine protease inhibitor with a molecular mass of 6106±2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed α/β-or α + β polypeptides. It was established that InhVJ is highly specific toward trypsin (K _i 2.49 × 10^?9 M) and α-chymotrypsin (K _i 2.17 × 10^?8 M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.     

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.