IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.     

In Vitro and in Vivo Evaluation of the Efficacy of Bovine Colostrum against Human Rotavirus Infection

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6–7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.     

ZINGIPAIN,A CYSTEINE PROTEASE FROM Zingiber ottensii VALETON RHIZOMES WITH ANTIPROLIFERATIVE ACTIVITIES AGAINST FUNGI AND HUMAN MALIGNANT CELL LINES

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH₄)₂SO₄ precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (～13.8 and ～15.2 kD) subunits or these two together with a larger (～32.5 kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)–mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6 µg F50/0.3 cm² of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13 µg/mL (hepatoma cancer; HEP-G2) to 5.37 µg/mL (colon cancer; SW620). By periodic acid–Schiff's staining and phenol–sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30 ± 1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.     

Proteomic methods reveal cyclophilin a function as a host restriction factor against rotavirus infection

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl‐prolyl cis‐trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.     

Methyl lucidenate F isolated from the ethanol-soluble-acidic components of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> is a novel tyrosinase inhibitor

Tyrosinase is a key enzyme in the biosynthesis of melanin, and the use of inhibitors against tyrosinase can prevent hyperpigmentation by inhibiting enzymatic oxidation. However, the current use of tyrosine inhibitors is limited by their low activities and high toxicities. The aim of the present research was to develop novel whitening agents, or tyrosinase-targeted medicine, from a submerged culture of the fungus Ganoderma lucidum. Methyl lucidenate F was isolated from the ethanol-soluble-acidic components (ESACs) of G. lucidum, with the structure of ESACs elucidated via UV, LC-MS, and ¹³C-NMR spectral analysis. The tyrosinase inhibitory activity was measured using catechol as a substrate. Methyl lucidenate F displayed uncompetitive inhibition of the potato tyrosinase activity, for which Lineweaver-Burk plots revealed a maximum reaction rate (V _max) of 0.4367/min, Michaelis constant (K _m) of 6.765 mM and uncompetitive inhibition constant (K _i) of 19.22 μM. Meanwhile, methyl lucidenate F (tetra cyclic triterpenoid) exhibited high tyrosinase inhibitory activity, with an IC₅₀ of 32.23 μM. These results suggest that methyl lucidenate F may serve as a potential candidate for skin-whitening agents.     

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH₂) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3₁₀ helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a β-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pK_a value in solution. It is concluded that the local structure present in the random coil state of VP2(156–170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

The chemical composition,antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with Thai orchids

Some plant‐derived bioactive compounds produced by fungal endophytes have been proven to have antimicrobial and antioxidant activities. In this study, endophytic fungi were isolated from 20 orchid samples collected in northern Thailand from 12 genera of orchids. In total, 97 isolates were isolated from the leaves (44.3%), stems (40.2%) and flowers (15.5%) of the orchid samples. The antifungal activity was investigated of the endophytic isolates against the plant pathogenic fungi. The results showed that 13 endophytic isolates provided antifungal activities against Fusarium sp., Colletotrichum sp. and Curvularia sp. The endophyte CK F05‐5, which was isolated from the flower part of Dendrobium lindleyi, was chosen for further testing because it the highest level of antifungal activity against Fusarium sp. The isolate CK F05‐5 was identified as Fusarium oxysporum on the basis of its ITS sequences of 5.8 s rRNA, and phytochemical analysis revealed the presence of coumarins. The ethyl acetate extract of CK F05‐5 was examined for its total phenolic content and antioxidant activity using Folin–Ciocalteu's reagent and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging assay, respectively. The phenolic content was 160.51 mg of GAE/g of extract, and the free radical scavenging activity was 89.61 µg/ml at the half maximal inhibitory concentration (IC₅₀). The antimutagenic potential of the ethyl acetate extract of CK F05‐5 against Trp‐P‐1 mutagenic substances was determined using the Ames test which revealed that the extract of CK F05‐5 at 10 mg/plate had the highest antimutagenic activity against Trp‐P‐1 (51.2%) and 39.6% for strains TA98 and TA100, respectively. The active compounds present in the acetate extract of CK F05‐5 were examined using GC‐MS analysis, which displayed the presence of gibepyrone A, pyrrolo [1, 2‐a] pyrazine‐1, 4‐dione, hexahydro‐3‐(2‐methylpropyl) and indoleacetic acid as major components. Based on the results, this endophytic fungus contains various bioactive components that have various biological activities. This useful information could help in producing potentially valuable and novel pharmaceutical products.     

Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease A subunit

Epitope vaccine based on urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In this study, we constructed an epitope vaccine with mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA_183-203) of H. pylori urease A subunit named CTB-UA. The CTB-UA fusion protein was expressed in Escherichia coli, and the purified protein was used for intraperitoneal immunization experiments in BALB/c mice. The experimental results indicated that anti-CTB-UA antibody could recognize both H. pylori urease A subunit (UreA) and urease B subunit (UreB). Besides, the CTB-UA epitope vaccine had good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies which showed effectively inhibitory effect on the enzymatic activity of H. pylori urease. CTB-UA is a promising molecule to be investigated as H. pylori vaccine antigen candidate.     

Protein glycation inhibitory activities of Lawsonia inermis and its active principles

The protein glycation inhibitory activity of ethanolic extract of Lawsonia inermis (henna) plant tissues was evaluated in vitro using the model system of bovine serum albumin and glucose. Protein oxidation and glycation are posttranslational modifications that are implicated in the pathological development of many age-related disease processes. This study investigated the effects of Lawsonia inermis ethanolic extract and its components, on protein damage induced by a free radical generator in in vitro assay system. We found that alcoholic extract of Lawsonia inermis can effectively protect against protein damage and showed that its action is mainly due to Lawsone. In addition, the presence of gallic acid also plays an important role in the protective activity against protein oxidation and glycation. Two known compounds, namely, Lawsone and gallic acid previously isolated from this plant were subjected to glycation bioassay for the first time. It was found that the alcoholic extract, lawsone (1) and gallic acid (2) showed significant inhibition of Advanced Glycated End Products (AGEs) formation and exhibit 77.95%, 79.10% and 66.98% inhibition at a concentration of 1500 μg/mL, 1000 μg/mL and 1000 μM respectively. Lawsonia inermis, compounds 1 and 2 were found to be glycation inhibitors with IC₅₀ 82.06 ± 0.13 μg/mL, 67.42 ± 1.46 μM and 401.7 ± 6. 23 μM respectively. This is the first report on the glycation activity of these compounds and alcoholic extract of Lawsonia inermis.     

Oral Passive Immunization Effect of Anti-Human Rotavirus IgY and Its Behavior against Proteolytic Enzymes

The neutralization titer of anti-human rotavirus (HRV) IgY was completely inactivated by pepsin at pH 2.0. However, it was not significantly affected by trypsin or chymotrypsin under certain conditions. The immunological activity of the IgY was observed in the intestine of suckling mice for 2 h after oral administration and the activity rapidly decreased thereafter. The effects of oral supply of IgY were thus estimated for HRV-induced diarrhea in suckling mice and it was found that a previous supply of the IgY, (1 h before HRV infection) completely prevented the HRV-induced diarrhea. The preventive effect was decreased as the time gap between IgY administration and HRV infection was longer. However, the oral supply of the IgY within 24h after HRV infection was still effective and decreased the incidence of HRV diarrhea in suckling mice.     

Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells

Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection. We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC), to HeLa cells. Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens.     

1-Methyladenine inhibits adenylate cyclase in starfish oocytes

Summary

The effect of 1-methyladenine (1-MeA) on adenylate cyclase (AC) basal activity and on preliminary stimulated AC activity was investigated in oocyte membrane preparations of the starfish Aphelasterias japonica. 1-MeA inhibited the membrane-bound AC activity both after its addition to intact oocytes and in cell-free experiments. GTP did not affect AC activity but it intensified the inhibitory effect of 1-MeA on AC activity. Sodium fluoride (F″) stimulated the oocyte AC (8 fold), while 1-MeA significantly reduced F″-stimulated activity. Manganese (MnCl₂, 5mM) stimulated AC (150 fold), but 1-MeA did not reduce Mn²⁺-stimulated activity. However, Mn²⁺-stimulated AC activity was inhibited by 1-MeA in the presence of MgCl₂. Forskolin stimulated AC activity (7 fold) and 1-MeA had no effect on AC. Thus, the inhibitory effect of 1-MeA on stimulated AC activity is displayed only after stimulation of the regulatory AC subunit. We suggest that 1-MeA inhibits the oocyte AC acting via inhibitory regulatory Gi protein of AC.     

Productivity and Some Properties of Egg Yolk Antibody (IgY) against Human Rotavirus Compared with Rabbit IgG

Productivity and some properties of anti-Human Rotavirus (HRV) hen egg yolk antibody (IgY) were compared with those of anti-HRV rabbit serum antibody (IgG). The hens immunized with HRV (Wa strain, serotype 1 and Mo strain, serotype 3) were found to continuously to lay eggs without any change in the egg laying rate and the yolk of the eggs laid over a year showed a high level of neutralization titer against HRV. The production of anti-HRV IgY by a hen (one year) was at least 15 times (anti-Wa) and 120 times (anti-Mo) more effective than those by an immunized rabbit in the neutralization titer of the antibodies.

The stability of anti-HRV IgY at temperature above 70°C and low pH 2–3 was less than that of anti-HRV rabbit IgG. The temperature corresponding to the maximum of denaturation endotherm (T_max) of IgY was 73.9°C while that of rabbit IgG was 77.0°C in the analysis by differential scanning calorimetry. This discrepancy in heat and acidic pH stability found between the two antibodies as discussed with regard to their protein structures.     

Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

     

Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations

Summary Fast protein liquid chromatography (FPLC) was used to characterize a commercial cellulase preparation (Celluclast 1.5L, Novozymes) in relation to its protein profile and activity against hydroxyethylcellulose (HEC) and other substrates. Co-elution of CBHII (Cel 6A) with other enzyme components of the cellulase system was characterized by immunochemical assays using monoclonal antibodies, whereas the occurrence of EGII (Cel 5A) was assessed based on its ability to cleave the heterosidic bond of 4-methylumbellyferyl-β-d-cellotrioside (MUmbG₃). The main cellulase constituents of Celluclast 1.5L were pooled into six fractions containing EGII (Cel 5A) and EGIII (Cel 12A) (F1), EGII and CBHII (Cel 6A) (F2), CBHII and EGI (Cel 7B) (F3), EGI (F4), and CBHI (Cel 7A) (F5). The occurrence of CBHI core protein within the CBHI fraction of the FPLC profile was determined by hydrophobic interaction chromatography. Using this method, we were able to demonstrate that the batch of Celluclast 1.5L used in this study contained 10.9–18.8% of CBHI as its corresponding free core protein.     

Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.     

Essential‐Oil and Fatty‐Acid Composition,and Antioxidant Activity of Extracts of Ficaria kochii

The essential‐oil and fatty‐acid composition of the aerial parts of Ficaria kochii (Ledeb .) Iranshahr & Rech .f. native to Iran, and the antioxidant activity of various extracts of this plant were examined. The study by GC‐FID and GC/MS analysis of the essential oil resulted in the identification of 61 compounds, representing 86.01% of the total oil composition. Phytol (10.49%), farnesol (7.72%), methyl linoleate (5.57%), and α‐farnesene (4.96%) were the main components. The fatty‐acid composition of the aerial parts of F. kochii was also analyzed by GC/MS. The major components were palmitic acid (25.9%), linolenic acid (25.3%), and linoleic acid (17.5%). Polyunsaturated fatty acids (PUFAs) were found in higher amounts than saturated fatty acids. The possible antioxidant activity of various extracts (prepared by using solvents with different polarity) of the F. kochii aerial parts was evaluated by screening for their 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical‐scavenging activity, Fe^III‐reducing power, total antioxidant activity, and inhibitory activity in the linoleic acid‐peroxidation system. H₂O proved to be the most efficient solvent for the extraction of antioxidants, as the H₂O extract contained the highest amount of phenolic compounds (2.78±0.23 GAE/g dry matter) and also exhibited the strongest antioxidant capacity in all the assays used. The results of the present investigation demonstrated that the aerial parts of F. kochii can be used as natural and safe nutrition supplement in place of synthetic ones.     

Derivatives of Holomycin and Cyclopropaneacetic Acid from Streptomyces sp. DT‐A37

On the basis of the one strain–many compounds strategy, five compounds including two new holomycin derivatives 2 – 3 , two new cyclopropaneacetic acid derivatives 4 – 5 , together with one known compound holomycin ( 1 ) were isolated from a marine‐derived bacterium Streptomyces sp. DT‐A37. Their structures were elucidated using NMR and HR‐ESI‐MS analyses. All these compounds were evaluated for their antimicrobial activity, cytotoxic activity, and inhibitory activity against BRD4 protein. Compound 1 exhibited potent cytotoxicity against H1975 cells with IC₅₀ value of 1 μm , and its minimal inhibitory concentration values against Escherichia coli and Staphylococcus aureus were both 64 μm .