In vitro efficacies of oils, silicas and plant preparations against the poultry red mite Dermanyssus gallinae

The aim of this study was to test the effectiveness of physically acting substances (oils and silicas) and plant preparations for the control of the poultry red mite Dermanyssus gallinae (De Geer 1778). Reproduction and survival of fed D. gallinae females were evaluated in vitro for a total of 168 h using the “area under the survival curve” (AUC) to compare survival of the mites between treatments. Four oils (two plant oils, one petroleum spray oil and diesel), one soap, three silicas (one synthetic amorphous silica, one diatomaceous earth (DE) and one DE with 2% pyrethrum extract) and seven plant preparations (derived from Chrysanthemum cineariaefolium, Allium sativum, Tanacetum vulgare, Yucca schidigera, Quillaja saponaria, Dryopteris filix-mas, and Thuja occidentalis) were tested at various concentrations. All the oils, diesel and soap significantly reduced D. gallinae survival. All silicas tested inhibited reproduction. DE significantly reduced mite survival, but amorphous silica was less effective in vitro. Except for pure A. sativum juice and the highest concentration of C. cineariaefolium extract, the plant preparations tested resulted in statistically insignificant control of D. gallinae.     

Whom do the sparrows follow? The effect of kinship on social preference in house sparrow flocks

Non-aggressive social interactions between group-mates, e.g. maintenance of spatial proximity or activity synchrony are basic elements of a species’ social structure, and were found to be associated with important fitness consequences in group-living animals. In the establishment of such affiliative relationships, kinship has often been identified as one of the key predictors, but this has rarely been studied in simple social groups such as flocks of gregarious birds. In this study we investigated whether kinship affects social preference, as measured by the tendency to associate with others during various social activities, in captive house sparrow (Passer domesticus) flocks where birds could interact with differently related flock-mates. We found that preference between flock-mates was correlated with familiarity from early nestling period: same-brood siblings followed their sib initiating new activities more often than non-sib birds. The strength of association between birds also tended to correlate with genetic relatedness, but this was mainly due to the effect of siblings’ affiliation. Thus we concluded that house sparrows prefer the company of their siblings during social activities even well after fledging, which may facilitate kin-biased behaviours.     

Synthesis and optical resolution of 1‐[(3‐carboxy‐1,1′‐biphenyl)‐2‐yl]‐1H‐pyrrole‐2‐carboxylic acid

Site selective mono‐ and dimetalation methods have been developed for the functionalization of 1‐[(1,1′‐biphenyl)‐2‐yl]‐1H‐pyrrole. Optical resolution of the prepared 1‐[(3‐carboxy‐1,1′‐biphenyl)‐2‐yl]pyrrole‐2‐carboxylic acid provided new atropisomeric 1‐arylpyrrole derivatives. The absolute configuration of the pure dicarboxylic acid enantiomers was determined by single crystal X‐ray diffraction and CD spectroscopy. Chirality 2009. © 2009 Wiley‐Liss, Inc.     

Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice

The present work evaluated the anxiolytic activity of an aqueous extract of Apocynum venetum L. (Apocynaceae) and bioguided its fractionation using the elevated plus maze (EPM) in mice as a model of anxiety. A single treatment of AV extract markedly increased the percentage time spent on the open arms of the EPM in two distinct concentration ranges of 22.5–30 and 100–125 mg/kg p.o., respectively, indicating a putative anxiolytic-like activity. Fractions showing anxiolytic effects in concentrations equal to 30 or 125 mg/kg of whole extract were antagonized using the benzodiazepine antagonist flumazenil (3 mg/kg i.p.) or the 5-HT_1A receptor antagonist WAY-100635 (0.5 mg/kg i.p.). All active fractions in a concentration equal to 125 mg/kg were effectively blocked by the benzodiazepine antagonist flumazenil, while the anxiolytic activities of fractions in the lower dose equivalent to 30 mg/kg of whole extract were inhibited by the 5-HT_1A receptor antagonist WAY-100635. Through further separation of AV fractions it was possible to isolate and characterize the flavonol kaempferol which showed an anxiolytic-like activity in concentrations from 0.02 to 1.0 mg/kg p.o. The anxiolytic activity of kaempferol was partially antagonized by concomitant administration of flumazenil, but not by WAY-100635. In conclusion, our study clearly demonstrates that AV extract possesses anxiolytic-like activity and that at least one of its flavonoids, kaempferol, can elicit the same kind of neuropharmacological activity.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer’s kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.     

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer’s kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.     

A preliminary phylogeny of the 'didymocarpoid Gesneriaceae' based on three molecular data sets: Incongruence with available tribal classifications

The 'didymocarpoid Gesneriaceae' (traditional subfam. Cyrtandroideae excluding Epithemateae) are the largest group of Old World Gesneriaceae, comprising 85 genera and 1800 species. We attempt to resolve their hitherto poorly understood generic relationships using three molecular markers on 145 species, of which 128 belong to didymocarpoid Gesneriaceae. Our analyses demonstrate that consistent topological relationships can be retrieved from data sets with missing data using subsamples and different combinations of gene sequences. We show that all available classifications in Old World Gesneriaceae are artificial and do not reflect natural relationships. At the base of the didymocarpoids are grades of clades comprising isolated genera and small groups from Asia and Europe. These are followed by a clade comprising the African and Madagascan genera. The remaining clades represent the advanced Asiatic and Malesian genera. They include a major group with mostly twisted capsules. The much larger group of remaining genera comprises exclusively genera with straight capsules and the huge genus Cyrtandra with indehiscent fruits. Several genera such as Briggsia, Henckelia, and Chirita are not monophyletic; Chirita is even distributed throughout five clades. This degree of incongruence between molecular phylogenies, traditional classifications, and generic delimitations indicates the problems with classifications based on, sometimes a single, morphological characters.     

��-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of α-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58^IPK promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.To ensure cellular homeostasis and to preclude toxic effects of aberrant protein conformers quality control mechanisms have evolved to recognize and degrade non-functional and misfolded proteins. In the cytosol the ubiquitin-proteasome system is the main pathway for regulated protein turnover (for reviews, see Refs. 1–3). Moreover the proteasome is part of a quality control system designated endoplasmic reticulum (ER)⁵-associated degradation (ERAD), which mediates post-translational degradation of non-native proteins generated in the ER. ERAD is the primary response to eliminate non-native ER proteins. It involves recognition of non-native polypeptides by ER-resident proteins and retrograde transport to the cytosol where proteasomal degradation occurs (for reviews, see Refs. 4–6). In case ERAD substrates accumulate in the ER lumen intracellular signaling pathways are induced that are collectively called the unfolded protein response (for reviews, see Refs. 7 and 8). Recently a preemptive, co-translocational quality control pathway was described that operates before translocation into the ER is completed (9, 10). Regulated translocation could act as an early quality control step to prevent an overload of the ER with non-native proteins. This regulation relies on an interplay between intrinsic features of the polypeptides and accessory factors able to modulate the translocation efficiency (for a review, see Ref. 11). Although numerous factors are known to be involved in ERAD less is known about mediators of the preemptive, co-translocational quality control pathway. In one study p58^IPK was identified as a key regulator (9), whereas in another it was shown that ER translocation during conditions of acute ER stress is controlled by the signal peptide (10). Indeed the signal peptide is an important intrinsic determinant. Although an exceptionally diverse set of sequences can target proteins for ER import it has been demonstrated that the translocation efficiency is modulated in a signal peptide sequence-specific manner (for reviews, see Refs. 12–14). Another attractive candidate for an intrinsic factor to regulate translocation is the folding state of the nascent polypeptide chain. Formation of secondary structure occurs already in the ribosome exit tunnel (15–18). Moreover it was shown that the polypeptide structure within the ribosomal exit tunnel can modulate translocation of distal parts of the nascent chain (19).The mammalian prion protein (PrP) is a suitable model protein to study whether formation of secondary structure could modulate translocation efficiency because it has an intriguing modular composition: the N-terminal domain spanning 120 amino acids is flexibly disordered followed by a highly structured C-terminal domain of ∼110 amino acids. This autonomously folding domain contains three α-helical regions and a short, two-stranded β-sheet (20–22). Notably folding of the C-terminal domain is one of the most rapid folding reactions measured in vitro (23). Interestingly previous studies indicated that the C-terminal folded domain of PrP is necessary and sufficient to promote ER import. In cultured cells and neurons of transgenic animals PrP-S230X (also known as PrPΔGPI or GPI⁻PrP), a mutant devoid of the C-terminal glycosylphosphatidylinositol (GPI) anchor signal peptide, is efficiently imported into the ER and secreted (24–27). However, by deleting parts of the α-helical domains located in the C-terminal domain a fraction is directed to proteasomal degradation in the cytosol (28).We have now analyzed the underlying mechanisms of this impaired ER import and show that ER-targeted proteins require a certain amount of structured domains to be imported into the ER. In addition, our study indicates that extended unstructured domains are signals for a preemptive/co-translocational degradation pathway.