Immunochemical characterization of rapid and slowly released allergens from the pollen of Parthenium hysterophorus

Allergens from the pollen of Parthenium hysterophorus (American feverfew), responsible for high incidence of allergic rhinitis, were found by immunoprint analysis to be localized on the surface of the pollen grains. The allergens were released very rapidly when extracted in vitro. The allergenic activity of the rapid (10 s) and slowly (20 h) released pollen proteins was comparable by in vivo skin test and ELISA inhibition assay. The isoelectric focusing patterns of the rapid and slowly released proteins, were also identical. SDS-PAGE and Western blot analysis revealed that all the major pollen allergens with molecular weight 14, 28, 31, 37 and 45 kDa were eluted within 10 s of extraction. Periodate-Schiff staining showed that the 28, 31 and 45 kDa components of the pollen extract are glycoproteins. The pollen allergens released after different periods of extraction lost 75% of IgE binding activity when subjected to in situ sodium m-periodate oxidation under controlled conditions, while 80% of the allergenic activity was still retained after extensive proteolysis. Our results support the clinical observation of a rapid onset of symptoms of allergic rhinitis in patients sensitive to Parthenium pollen, mediated predominantly by glycoproteins.

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

Background: Bryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle. Methods: A chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step-gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells. Results: Results showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 mu g/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50: 50) and showed IC50 at 91 mu g/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A. Conclusions: Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

Genetic evidence of a strong functional constraint of neurotrypsin during primate evolution

Neurotrypsin is one of the extra-cellular serine proteases that are predominantly expressed in the brain and involved in neuronal development and function. Mutations in humans are associated with autosomal recessive non-syndromic mental retardation (MR). We studied the molecular evolution of neurotrypsin by sequencing the coding region of neurotrypsin in 11 representative non-human primate species covering great apes, lesser apes, Old World monkeys and New World monkeys. Our results demonstrated a strong functional constraint of neurotrypsin that was caused by strong purifying selection during primate evolution, an implication of an essential functional role of neurotrypsin in primate cognition. Further analysis indicated that the purifying selection was in fact acting on the SRCR domains of neurotrypsin, which mediate the binding activity of neurotrypsin to cell surface or extracellular proteins. In addition, by comparing primates with three other mammalian orders, we demonstrated that the absence of the first copy of the SRCR domain (exon 2 and 3) in mouse and rat was due to the deletion of this segment in the murine lineage. Copyright (C) 2005 S. Karger AG, Basel.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Construction and characterization of a novel recombinant single-chain variable fragment antibody against White Spot Syndrome Virus from shrimp

An antibody phage display library against White Spot Syndrome Virus (WSSV) was constructed. After four rounds of panning against WSSV, 192 out of 480 clones displayed WSSV binding activity. One of the positive clones, designated A1, had relatively higher activity specifically binding to WSSV A1-soluble, single-chain fragment variable (scFv) antibody has an affinity constant (K-aff) of 2.02 +/- 0.42 x 10(9) M-1. Dot blot assays showed that A1-soluble scFv could detect WSSV directly from shrimp hemolymph after 24-h feeding infection by WSSV. A1 scFv has potential for the development of a cheap, simple and sensitive diagnostic kit for WSSV in the field. (C) 2003 Elsevier Science B.V. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis

A natural lectin from the plasma of the shrimp Fenneropenaeus chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75 degrees C and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Distinct functions of POT1 at telomeres.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.

Involvement of Rel/Nuclear Factor-κB Transcription Factors in Keratinocyte Senescence

After a finite doubling number, normal cells become senescent, i.e. nonproliferating and apoptosis resistant. Because Rel/nuclear factor (NF)-κB transcription factors regulate both proliferation and apoptosis, we have investigated their involvement in senescence. cRel overexpression in young normal keratinocytes results in premature senescence, as defined by proliferation blockage, apoptosis resistance, enlargement, and appearance of senescence-associated β-galactosidase (SA-β-Gal) activity. Normal senescent keratinocytes display a greater endogenous Rel/NF-κB DNA binding activity than young cells; inhibiting this activity in presenescent cells decreases the number of cells expressing the SA-β-Gal marker. Normal senescent keratinocytes and cRel-induced premature senescent keratinocytes overexpressed manganese superoxide dismutase (MnSOD), a redox enzyme encoded by a Rel/NF-κB target gene. MnSOD transforms the toxic O2.- into H2O2, whereas catalase and glutathione peroxidase convert H2O2 into H2O. Neither catalase nor glutathione peroxidase is up-regulated during cRel-induced premature senescence or during normal senescence, suggesting that H 2O2 accumulates. Quenching H2O2 by catalase delays the occurrence of both normal and premature cRel-induced senescence. Conversely, adding a nontoxic dose of H2O2 to the culture medium of young normal keratinocytes induces a premature senescence-like state. All these results indicate that Rel/NF-κB factors could take part in the occurrence of senescence by generating an oxidative stress via the induction of MnSOD.

Functional characterization of a Kar3/Vik1-like Kinesin-14 heterodimer from the filamentous multinucleate fungus Ashbya gossypii

Kinesins are motor proteins that convert chemical energy from ATP hydrolysis into mechanical energy used to generate force along microtubules, transporting organelles, vesicles, and proteins within the cell. Kar3 kinesins are microtubule minus-end-directed motors with pleiotropic functions in mating and mitosis of budding and fission yeast. In Saccharomyces cerevisiae, Kar3 is multifunctionalized by two non-catalytic companion proteins, Vik1 and Cik1. A Kar3-like kinesin and a single Vik1/Cik1 ortholog are also expressed by the filamentous fungus Ashbya gossypii, which exhibits different nuclear movement challenges and unique microtubule dynamics from its yeast relatives. We hypothesized that these differences in A. gossypii physiology could translate into interesting and novel differences in its versions of Kar3 and Vik1/Cik1. Presented here is a structural and functional analysis of recombinantly expressed and purified forms of these motor proteins. Compared to the previously published S. cerevisiae Kar3 motor domain structure (ScKar3MD), AgKar3MD displays differences in the conformation of the ATPase pocket. Perhaps it is not surprising then that we observed the maximal microtubule-stimulated ATPase rate (kcat) of AgKar3MD to be approximately 3-fold slower than ScKar3MD, and that the affinity of AgKar3MD for microtubules (Kd,MT) was lower than ScKar3MD. This may suggest that elements that compose the ATPase pocket and that participate in conformational changes required for efficient ATP hydrolysis or products release work differently for AgKar3 and ScKar3. There are also subtle structural differences in the disposition of the secondary structural elements in the small lobe (B1a, B1b, and B1c) at the edge of the motor domain of AgKar3 that may reflect the enhanced microtubule-depolymerization activity that we observed for this motor, or they could relate to its interactions with a different regulatory companion protein than its budding yeast counterpart. Although we were unable to gain experimentally determined high-resolution information of AgVik1, the results of Phyre2-based bioinformatics analyses may provide a structural explanation for the limited microtubule-binding activity we observed. These and other fundamental differences in AgKar3/Vik1 could explain divergent functionalities from the ScKar3/Vik1 and ScKar3/Cik1 motor assemblies.

The role of advanced glycation end products in retinal microvascular leukostasis

PURPOSE: A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature. METHODS: Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA: In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown. RESULTS: Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P <0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P <0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P <0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P <0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P <0.001). CONCLUSIONS: AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.