Interaction of Axl receptor tyrosine kinase with C1-TEN,a novel C1 domain-containing protein with homology to tensin

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.     

Methylparaben induces malformations and alterations on apoptosis,oxidant–antioxidant status,ccnd1 and myca expressions in zebrafish embryos

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant–antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT‐PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione‐S‐transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch‐evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research.     

Ringworm in rabbit due to Trichophyton quinckeanum

Summary Trichophyton quinckeanum was isolated from a spontaneous infection in rabbit. The hairs were also invaded by the fungus, exibiting a yellowish fluorescence in Wood's light. White mice inoculation of the isolate produced typical scutula with hair penetration fluorescing in green colour. The type of animal hair invasion is also discussed. The morphologic features ofTr. quinckeanum, together with its ability of producing scutula while inoculating the white mice, must be emphasized, when proving its separate identity.     

Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.     

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 × 10⁶ sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5 mM (55.5 ± 7.38%) and cysteine 10 mM (48 ± 5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99 ± 0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Effects of bovine milk lactoperoxidase system on some bacteria

Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG-50-H⁺ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified 20.45-fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and a single band was observed. K _m was 0.25 mM at 20°C, V _max value was 7.95 μmol/ml min at 20°C (pH 6.0). Antibacterial study was done by disk diffusion method of Kirby-Bauer using Mueller-Hinton agar medium with slight modification. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate-100 mM H₂O₂ medium for some bacteria (Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces violaceusniger, Vibrio cholerae-nonO1) respectively, and compared with well known antibacterial substances (levofloxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO-100 mM thiocyanate-100 mM H₂O₂ system was proposed as an effective agent against many factors causing several diseases.     

Influence of growth conditions on the nisin production of bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright © 2014 John Wiley & Sons, Ltd.