Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part II: Studies in the SDS micelle.

There is much evidence to support the hypothesis that lipids play a role in the interaction of peptide hormones with their membrane receptors. This interaction through change of peptide conformation can facilitate the entry of the hormone into the microenvironment of the receptor. In the present study we have examined the interaction of vasopressin and mesotocin with a lipid-sodium dodecylsulfate (SDS) micelle-using 2D nuclear magnetic resonance (NMR) and theoretical methods. Solution structures of two hormones in solution with SDS were established using the nuclear Overhauser effect (NOE) and the (3)J(NHHalpha) couplings. The amino acid sequences of these peptides are: c[C(1)-Y(2)-F(3)-Q(4)-N(5)-C(6)]-P(7)-R(8)-G(9)-NH(2) ([Arg(8)]vasopressin, AVP) and c[C(1)-Y(2)-I(3)-Q(4)-N(5)-C(6)]-P(7)-I(8)-G(9)-NH(2) (MT). Each of the peptides was found to occur as one stable conformation. AVP adopts the cis configuration on the Cys(1)-Tyr(2) peptide bond, a finding not reported so far. The three-dimensional structures of the two peptides studied were determined by a method that consisted of time-averaged molecular dynamics in an explicit SDS micelle with the parm99 force field in AMBER8.0 package. All calculated structures of the studied peptides form beta-turns in their cyclic parts. The C-terminal fragment of MT is bent, whereas that of AVP is extended.     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates

p97/VCP associated with Ufd1-Npl4 is considered a key player in ER-associated degradation (ERAD). RNA interference (RNAi) of one component of the Ufd1-Npl4 heterodimer destabilizes the VCP-Ufd1-Npl4 complex inducing proteasome-dependent degradation of the other component and releasing free VCP. In contrast to RNAi of VCP, RNAi of Ufd1 or Npl4 depleting approximately 90% of the VCP-Ufd1-Npl4 complexes does not induce unfolded protein response, indicating that the Ufd1-Npl4 dimer is not involved in the regulation of ER function by VCP. RNAi of Ufd1 or Npl4 is associated with a 2-fold increase in the levels of polyubiquitinated proteins, which form dispersed aggregates often associated with calnexin-positive structures. However, contrary to the effects of proteasome inhibition, RNAi of Ufd1 or Npl4 does not induce an accumulation of alpha-TCR and delta-CD3, two ERAD substrates overexpressed in HeLa cells. Instead, a 60-70% decrease in their levels is observed. The decrease in alpha-TCR levels is associated with a 50% decrease of its half-life. Upregulation of the putative channel forming protein, derlin-1, may contribute to the increased degradation of ERAD substrates. To explain our findings, we propose a model, where association of emerging ERAD substrates with VCP-Ufd1-Npl4 is not required for their degradation but has a regulatory role.     

Integrin alpha2beta1 mediates the anti-angiogenic and anti-tumor activities of angiocidin, a novel tumor-associated protein

We recently characterized an anti-tumor protein termed angiocidin. Here, we report that angiocidin may inhibit angiogenesis by binding collagen and its receptors. Angiocidin bound purified type I collagen and alpha2beta1 with high affinity. K562 cells expressing alpha2beta1 bound and adhered to angiocidin while K562 cells which only expressed alpha5beta1 integrin showed no binding and adhesion. Binding was specific since a neutralizing antibody against alpha2beta1 inhibited binding but antibodies against alpha5beta1 had no effect. Additionally, angiocidin co-localized with alpha2beta1 on K562 alpha2beta1 transfected cells, pancreatic cancer colo 357 cells, breast cancer MB-231 cells and human umbilical endothelial vein (HUVE) cells. In an alpha2beta1-dependent collagen gel angiogenesis assay, angiocidin showed potent inhibitory activity. We identified a 20-amino-acid amino terminal peptide of angiocidin that bound both alpha2beta1 and type I collagen. This peptide promoted alpha2beta1-dependent cell adhesion and inhibited tumor growth and angiogenesis. Taken together, these results are consistent with the conclusion that the anti-tumor activity of angiocidin arises from its ability to ligate collagen and alpha2beta1 on endothelial cells and tumor cells. Our results provide support for the concept that targeting matrix-cell interactions is a viable strategy for the development of anti-cancer therapeutics.     

Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin

Obtustatin is a potent and selective inhibitor of the alpha1beta1 integrin in vitro and of angiogenesis in vivo. It possesses an integrin recognition loop that harbors, in a lateral position, the inhibitory 21KTS23 motif. We report an analysis of the dynamics of the backbone and side-chain atoms of obtustatin by homonuclear NMR methods. Angular mobility has been calculated for 90 assigned cross-peaks from 22 off-resonance rotating frame nuclear Overhauser effect spectroscopy spectra recorded at three magnetic fields. Our results suggest that the integrin binding loop and the C-terminal tail display concerted motions, which can be interpreted by hinge effects. Among the integrin-binding motif, threonine 22 and serine 23 exhibit the lowest and the highest side-chain flexibility, respectively. It is noteworthy that the side chain of threonine 22 is not solvent-exposed, although based on synthetic peptides it appears to be the most critical residue for the inhibitory activity of obtustatin on the binding of integrin alpha1beta1 to collagen IV. Instead, the side chain of threonine 22 is oriented toward the loop center and hydrogen-bonded to residues Thr25 and Ser26. This network of interactions explains the restrained mobility of threonine 22 and suggests that its functional importance lies in maintaining the active conformation of the alpha1beta1 inhibitory loop.     

Laboratory trials to infect insects and nematodes by some acaropathogenic Hirsutella strains (Mycota: Clavicipitaceous anamorphs)

Laboratory assays have been carried out to artificially infect insect larvae of the birch bark-beetle (Scolytus ratzeburgi Jans.-Coleoptera, Scolytidae) and codling moth Cydia pomonella L. -Lepidoptera, Tortricidae) as well as the potato cyst nematode-Globodera rostochiensis Wollenweber, sugar beet nematode-Heterodera schachtii Schmidt and root-knot nematode-Meloidogyne hapla Chif (Nematoda, Heteroderidae), by the phialoconidia of some fungal species of the genus Hirsutella. From among four species tested on insects only H. nodulosa Petch infected about 20% of S. ratzeburgi larvae, whereas H. kirchneri (Rostrup) Minter, Brady et Hall, H. minnesotensis Chen, Liu et Chen, and H. rostrata Ba?azy et Wi?niewski did not affect insect larvae. Only single eggs of the root-knot nematode were infected by H. minnesotensis in the laboratory trials, whereas its larvae remained unaffected. No infection cases of the potato cyst nematode (G. rostochiensis) and sugar beet nematode eggs were obtained. Comparisons of DNA-ITS-region sequences of the investigated strains with GenBank data showed no differences between H. minnesotensis isolates from the nematodes Heterodera glycines Ichinohe and from tarsonemid mites (authors' isolate). A fragment of ITS 2 with the sequence characteristic only for H. minnesotensis was selected. Two cluster analyses indicated close similarity of this species to H. thompsonii as sister clades, but the latter appeared more heterogenous. Insect and mite pathogenic species H. nodulosa localizes close to specialized aphid pathogen H. aphidis, whereas the phytophagous mite pathogens H. kirchneri and H. gregis form a separate sister clade. Hirsutella rostrata does not show remarkable relations to the establishment of aforementioned groups. Interrelated considerations on the morphology, biology and DNA sequencing of investigated Hirsutella species state their identification more precisely and facilitate the establishment of systematic positions.     

Diversity of acaropathogenic fungi in Poland and other European countries

The occurrence, species diversity and some aspects of taxonomical affinity and host selectivity of acaropathogenic fungi associated with phytophagous, saprotrophic and predacious mites in Poland and other European countries were investigated on wild and cultivated plants, in insect feeding sites under the bark and in decayed wood. From among 33 species of fungi affecting mites only five species of Entomophthorales were separated and the most numerous were Neozygites floridana mostly on Tetranychus urticae, N. abacaridis on a few eriophyid species, and Conidiobolus coronatus attacking gamasid mites most frequently of the genus Dendrolaelaps. The most frequent mite pathogens occurring in mite communities on plants and in wood infested by insects were of the genus Hirsutella. Until now 13 of their form-species have been recognized in these habitats, but only H. kirchneri, H. necatrix and H. thompsonii (including its variety synnematosa) can be treated as exclusive oligophagous pathogens of phytophagous mites, though their potential host range seems to embrace only selected eriophyid or tarsonemid mites. Taxonomical differentiation of fungal strains was based on close morphological observations and molecular analysis of ITS region sequences. Two new species of acaropathogenic fungi were described in these studies. Hirsutella danubiensis sp. nov. was found in the tetranychid T. urticae, whereas H. vandergeesti sp. nov. affected phytoseiid mites of the genera Amblyseius, Neoseiulus, Seiulus and Typhlodromus, and the tarsonemid Tarsonemus lacustris.